Influence of type and level of dietary calcium on fluoride bioavailability in the rat

NUTRITION RESEARCH, Vol. 7, pp. 1073-1083, 1987 0271-5317/87 $3.00 + .00 Printed in the USA.

Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

INFLUENCE OF TYPE AND LEVEL OF DIETARY CALCIUM ON FLUORIDE BIOAVAILABILITY IN THE RAT 1 Florian L. Cerklewski,

Ph.D.

and James W. Ridlington,

Ph.D.

Oregon State University, College of Home Economics Department of Foods and Nutrition, Corvallis, OR 97331 ABSTRACT Two experiments were conducted with weanling rats fed a purified diet to determine the influence of dietary calcium (0.i, 0.2, 0.5, 1.0%) on fluoride bioavailability (2 or 10 ppm as sodium fluoride). The chemical form of calcium was the carbonate in experiment i and the gluconate in experiment 2. During each 6-week period the highest level of dietary calcium significantly reduced apparent fluoride absorption relative to controls (0.5% Ca), which was reflected in fluoride content of femur and most clearly in molar teeth, regardless of the solubility of calcium. When all levels of dietary calcium were considered, fecal fluoride excretion was found to be directly related to dietary calcium whereas urinary fluoride excretion was inversely related to calcium regardless of the solubility of calcium or the level of dietary fluoride. These effects, however, were markedly influenced by the fact that the amount of absorbed fluoride that was actually retained for incorporation into bones and teeth was directly related to dietary calcium level. Although high dietary calcium significantly depressed fluoride bioavailability during growth, it is equally clear that predictions about the magnitude of this relationship originating from gastric intubation studies have been greatly overstated. KEY WORDS:

Fluoride,

calcium,

bioavailability

INTRODUCTION The absorption of fluoride from foods is about 50-80% in contrast to essentially complete absorption from drinking water (1,2). The importance of this observation is that foods can make a significant contribution to total fluoride intake when foods are prepared with fluoridated water, especially for younger individuals where the need for fluoride is greatest, to develop bones and teeth (2). One of the food factors that has long been believed to influence fluoride absorption and utilization, defined as bioavailability (3), is the level of dietary calcium. Lawrenz and Mitchell (4), for example, demonstrated over 40 years ago that calcium added to the diet of rats as calcium phosphate dibasic would clearly reduce fluoride bioavailability from cryolite. Simultaneous administration of fluoride as sodium fluoride and calcium as the chloride by gastric intubation has also been shown to reduce absorption and skeletal uptake of fluoride in rats (5-7) at calcium-to-fluoride molar ratios ranging from 0.5 to 5.0. High dietary calcium (2%) has been shown to reduce fluoride absorption in rats given toxic doses of fluoride (8), and dietary calcium deficiency has been reported to enhance fluoride absorption (9,10). 1To whom reprint requests

should be addressed.

1073

1074

F.L. CERKLEWSKI and J.W, RIDLINGTON

In c o n t r a s t to these animal studies, Spencer et al. (ii) were u n a b l e to demonstrate a significant e f f e c t of c a l c i u m g l u c o n a t e on fecal fluoride e x c r e t i o n of adult h u m a n volunteers. A l t h o u g h these authors c o n c l u d e d that animal data cannot be e x t r a p o l a t e d to the h u m a n situation, two other h u m a n studies (12, 13) have clearly d e m o n s t r a t e d that supplemental oral c a l c i u m reduces fluoride a b s o r p t i o n based upon a change in p l a s m a fluoride concentration. In the study by Jowsey and Riggs (12), however, fluoride b i o a v a i l a b i l i t y was o n l y reduced to a level o n e - t h i r d that p r e d i c t e d by gastric intubation studies in rats (5-7) even at a m u c h higher c a l c i u m - t o - f l u o r i d e molar ratio. In the p r e s e n t study, our intent w a s to define the calcium and fluoride r e l a t i o n s h i p in growing rats using several indices of fluoride bioavailability. We specifically w i s h e d to determine if the m a g n i t u d e of the c a l c i u m and fluoride r e l a t i o n s h i p p r e d i c t e d by gastric i n t u b a t i o n studies could be o b s e r v e d u n d e r nutritional diet feeding conditions.

MATERIALS

A N D METHODS

E x p e r i m e n t 1 diets. The p u r p o s e of this study was to d e t e r m i n e the effect of calcium added to a c a l c i u m - d e f i c i e n t diet as the carbonate on apparent fluoride a b s o r p t i o n and r e t e n t i o n and fluoride u p t a k e by d e v e l o p i n g bone and m o l a r teeth. Diets were p r e p a r e d by adding reagent grade c a l c i u m carbonate and sodium fluoride, using glucose as a carrier, to a p u r i f i e d diet to p r o v i d e 0.i, 0,2, 0.5 (control) and 1.0% calcium w i t h either 2 or i0 p p m fluoride. The p u r i f i e d diet, which has been d e s c r i b e d in greater detail elsewhere (14), contained 15% v i t a m i n - f r e e casein, 0.3% DL-methionine, 4% cellulose powder, 5% v i t a m i n mixture, 2.785% m i n e r a l m i x t u r e (to p r o v i d e 1 g Ca/Kg diet), 5% corn oil, 15% cornstarch, and 52.92% dextrose. C o m p o s i t i o n of the v i t a m i n mixture has b e e n p r e v i o u s l y r e p o r t e d (14). The mineral m i x t u r e is supplied in grams/Kg diet: CaCOs, 2.497; Na2HP04, 15.083; K2HPO4, 3.408; KCI, 3.154; K2SO4, 1.818; MgCO 5 (26% Mg), 1.538; Z n C ~ , 0.0173; MnCO 5, 0.1109; CuCO 5, 0.0091; ferric citrate (18.42% Fe), 0.1901; KIOs, 0.0003; N a 2 S e O 5. 5H20 , 0.0003; CrK(S04) 2 9 12H20, 0.0192; NaF, 0.0044. Analysis of diet sample prior to the start of the study showed that the actual c o n c e n t r a t i o n of calcium was w i t h i n 3% of the calculated value. Actual c o n c e n t r a t i o n of fluoride was found to be + 0.i ppm for each c a l c u l a t e d fluoride level. E x p e r i m e n t 2 diets. The p u r p o s e of this study was to d e t e r m i n e the effect of d i e t a r y calcium as the w a t e r - s o l u b l e gluconate on fluoride bioavailability. The diet was identical to experiment 1 except that the mineral m i x t u r e was u s e d at 3.609% and dextrose was 52.09%. The source of calcium in the mineral m i x t u r e was reagent grade calcium g l u c o n a t e (9.31% Ca) at 10.741 g/Kg diet (I g C a / K g diet). C a l c i u m gluconate and sodium fluoride were added to the diet to p r o v i d e the calcium and fluoride levels stated for e x p e r i m e n t i. Procedures. In both studies, six m a l e o u t - b r e d S p r a g u e - D a w l e y albino rats (CRI:CD SD BR, Charles River Laboratories, Wilmington, MA), initial age and w e i g h t of 25 d and 60-70 g, respectively, were assigned to each of the 8 t r e a t m e n t diets (N=48). Each rat was individually housed, w i t h free access to the p o w d e r - t y p e diet and d i s t i l l e d - d e i o n i z e d water, as p r e v i o u s l y d e s c r i b e d (14). A five-day c o l l e c t i o n of feces and urine was m a d e for each rat during the third week of each study to estimate apparent fluoride a b s o r p t i o n (intake fecal) and r e t e n t i o n (intake - (fecal + urine)) (14). At the end of six weeks, rats were k i l l e d by d e c a p i t a t i o n after drawing blood from the abdominal aorta

CALCIUM AND FLUORINE INTERACTION

1075

under light sodium p e n t o b a r b i t a l anesthesia. Fluoride content of urine, ashed molars, u n a s h e d diet and feces was d e t e r m i n e d w i t h a fluoride combination electrode (14). C a l c i u m content of diet and femur was d e t e r m i n e d by atomic absorption s p e c t r o p h o t o m e t r y (Perkin-Elmer model no. 2380, Norwalk, CT). The statistical design for each study w a s a 2 X 4 factorial experiment w i t h six replicates p e r treatment (15). T r e a t m e n t effects were p a r t i t i o n e d into effects of fluoride, calcium and the i n t e r a c t i o n of the two factors if a significant F-value was found for treatment effects. Differences b e t w e e n p l a n n e d comparisons of m e a n s were tested by Fisher's least significant difference (FLSD) (15). Effects were c o n s i d e r e d to be s i g n i f i c a n t at P<0.05.

RESULTS Food Intake and Growth. Food intake and w e i g h t gain were e s s e n t i a l l y u n a f f e c t e d by d i f f e r e n c e s in type and level of dietary calcium and level of dietary fluoride (Table i). One e x c e p t i o n to this statement was the slower rate of growth o b s e r v e d in rats fed a diet c o n t a i n i n g 0.1% c a l c i u m as the carbonate and i0 p p m fluoride when c o m p a r e d to other groups. Fluoride E x c r e t i o n and Retention. Rats fed diets containing either 0.i or 0.2% calcium h a d an apparent fluoride a b s o r p t i o n that was nearly complete regardless of the chemical form of c a l c i u m or the d i e t a r y level of fluoride (Table 2). A p p a r e n t fluoride r e t e n t i o n in these same animals, however, was often, although not always, less than that o b s e r v e d in rats fed diets containing either 0.5% (controls) or 1.0% calcium. On the other hand, the lowest percent fluoride a b s o r p t i o n was found for rats fed diets containing 1.0% calcium in b o t h experiments. Percent fluoride r e t e n t i o n in these same animals was almost always similar to controls. In general, fecal fluoride e x c r e t i o n v a r i e d d i r e c t l y with d i e t a r y calcium level, w h e r e a s u r i n a r y fluoride e x c r e t i o n was inv e r s e l y related to calcium regardless of the chemical form of c a l c i u m or the level of dietary fluoride (Table 3). The amount of absorbed fluoride that was actually r e t a i n e d (biological v a l u e ) ( T a b l e 2) was essentially identical to e s t i m a t e d p e r c e n t apparent fluoride r e t e n t i o n for rats fed diets containing either 0.i or 0.2% calcium. Rats fed diets c o n t a i n i n g either 0.5 or 1.0% calcium, however, h a d a higher b i o l o g i c a l value for fluoride than for p e r c e n t fluoride retention. Biological value for fluoride was directly r e l a t e d to dietary calcium level in b o t h experiments. Fluoride and C a l c i u m C o n t e n t of Femur. B o t h the ash w e i g h t and calcium content of femur v a r i e d directly with dietary c a l c i u m level (Table 4). Fluoride c o n c e n t r a t i o n of femur was inversely r e l a t e d to the level of dietary calcium in both experiments. This r e l a t i o n s h i p b e t w e e n dietary c a l c i u m and femur fluoride was e s s e n t i a l l y m a i n t a i n e d in rats fed diets containing i0 p p m fluoride, even after correcting for d i f f e r e n c e s in ash w e i g h t by expressing results as ug fluoride per femur (Table 4). The e x c e p t i o n to t h i s statement was for rats fed diets containing 0.1% calcium. In rats fed diets containing 2 p p m fluoride, the effect of d i e t a r y calcium on total fluoride content of femur was only e v i d e n t when controls were c o m p a r e d to the high c a l c i u m group. Molar Fluoride. In contrast to femur, ash weights of second and third m o l a r s were u n a f f e c t e d by dietary c a l c i u m level (Table 5). For this reason molar fluoride has b e e n expressed only on a c o n c e n t r a t i o n basis. These results demonstrate that m o l a r uptake of fluoride was inversely related to dietary calcium regardless of the chemical form of calcium. C a l c i u m effects, however, were m o r e evident in rats fed diets c o n t a i n i n g i0 p p m fluoride c o m p a r e d to 2 p p m fluoride (P<0.001).

1076

F.L. CERKLEWSKIand J.W. RIDLINGTON

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DISCUSSION The results of our studies leave no doubt that the amount of dietary calcium ingested in an important factor influencing dietary fluoride b i o a v a i l a b i ~ ity during growth. Contrary to opinions expressed by others (11,12), we could find no reason to conclude that the solubility of calcium is important in this regard. In our studies, fluoride bioavailability w a s i n f l u e n c e d equally well by both a water-soluble, neutral form of dietary calcium (calcium gluconate) and a water-insoluble, alkaline form of calcium (calcium carbonate). Our results, as well as those from two human studies (12,13), however, indicate that the magnitude of the depressive effect of high calcium on fluoride bioavailability predicted by gastric intubation studies (5-7) has been greatly overestimated. In the study by Weddle and Muhler (5), for example, a calcium-to-fluoride molar ratio of five was found to depress femur fluoride by 74% when both factors were administered by gastric i n t u b a t i o n . Jowsey and Riggs (12), on the other hand, could only demonstrate a 22% reduction in fluoride bioavailability in human volunteers given oral calcium despite a calcium-to-fluoride molar ratio of 28. In the present study, when both calcium and fluoride were part of the diet, fluoride absorption was only depressed 15-26% despite a calcium-tofluoride molar ratio of 476. We therefore conclude that human and rat studies do agree that calcium depresses fluoride bioavailability when both factors are simultaneously present in the diet. The effect, however, is nowhere near the dire predictions originating from gastric intubation studies. Our results also demonstrated that a full understanding of the relationship between calcium and fluoride requires the use of several indices of fluoride bioavailability in the same test subject. Fluoride uptake by developing bone and teeth, for example, was poor in rats fed calcium-deficient diets relative to controls despite very efficient apparent fluoride absorption. Poor mineralization of bone was accompanied by elevated urinary fluoride excretion in these calcium-deficient animals. Most of total body fluoride is in bone (16) and the utilization of fluoride has been shown to be important for strengthening the mineral apatite structure of bone (17). Furthermore, rats fed a diet containing twice the calcium recommended for normal growth (18) tended to be the most efficient in retaining absorbed fluoride for incorporation into developing bone and molar teeth. This calcium effect therefore blunted the depressive effect of high dietary calcium on apparent fluoride absorption with regard to fluoride uptake by these two tissues. With regards to a mechanism to explain the depressive effect of high dietary calcium (1.0%) on fluoride bioavailability relative to controls (0.5%), our results are consistent with gastric intubation studies (5-7) which indicated that high calcium reduces fluoride absorption by way of insoluble complex formation. In our studies, for example, comparison of the 0.5 and 1.0% calcium groups, where calcium status was normal, showed little or no effect of calcium on apparent fluoride retention. Overall, we conclude that the relationship between calcium and fluoride would only be relevant to situations where both factors are simultaneously ingested. Calcium and fluoride, for example, can occur together in food since food prepared with fluoridated water can make a significant contribution to total fluoride intake, especially for younger age groups where the need for fluoride to strengthen developing bone and teeth is greatest (2). The United States is the only country where fluoridation has been sufficiently widespread to influence fluoride content of food (19). The depressive effect of calcium on fluoride bioavailability could also influence the amount of fluoride necessary to stabilize bone loss in patients suffering from osteoporosis as suggested by Jowsey and Riggs (12). Indiscriminate use of calcium supplements for perceived benefits may also provide an undesirable adverse effect of calcium on

1082

F.L. CERKLEWSKIand J.W. RIDLINGTON

fluoride

bioavailability

for the general

population.

ACKNOWLEDGEMENT We thank J.H. Baek and J. Song for their technical assistance. This r e s e a r c h was supported by N I H (NIDR) DE 05628 and the O r e g o n A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , t e c h n i c a l p a p e r no. 8248.

REFERENCES i.

Singer L, Ophaug RH. Fluoride. Present Knowledge in Nutrition, Foundation, 1984: 538-547.

2.

Rao GS. Dietary intake and b i o a v a i l a b i l i t y of fluoride. In: Darby WF, ed. Annual Review of Nutrition, Vol. 4 Palo Alto CA: A n n u a l Reviews Inc., 1984: 115-136.

3.

O'Dell

4.

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