Archs oral Bid. Vol. 36, No. 4, pp. 291-297, 1991 Printed in Great Britain. All rights reserved
0003-9969/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
FLUORI:DE, CALCIUM AND PHOSPHORUS METABOLISM IN THE RAT: COMPARISON OF ‘NATURAL IYNGREDIENT’ WITH SEMIPURIFIED DIETS G. M. WHITFORD Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, U.S.A. (Received 30 March 1990; accepted 14 November 1990)
Summary-Three groups of weanling female rats were fed different, commercially available, ‘natural ingredient’ diets containing 12,28 or 45 parts/lo6 F, mainly as bone meal, for six weeks. Two other groups were fed a low-fluoride (0.76 parts/106) semipurified diet. They received fluoride doses, either in the drinking water or by daily intraperitoneal injection, which were approximately equal to the average dose of the other three groups. Rats on the ‘natural ingredient’ diets ingested more food and water and excreted more faeces and urine, effects which were attributed to the higher amounts of dietary fibre, Na, K and Cl. Thus, at any given concentration of fluoride in the food or water, the level of fluoride ingestion and the ensuing efFects would be influenced by the type of diet used. The values for fractional fluoride absorption (45-49%) and retention (38-47%) were similar among the groups given ‘natural ingredient’ diets. In the groups given semipurified diet, the corresponding values were about twice as high with the exception that fractional absorption was negative (-41%) in the injected group, which indicated net intestinal secretion of fluoride. Fluoride balances and tissue concentrations were highest in the groups fed the semipurified diet, even though the level of intake was not always higher. The fractional values for calcium and phosphorus absorption (41-51%) and retention (33-43%) were also similar among the groups given ‘natural ingredient’ diets. The corresponding values were about twice as high in the groups fed the semipurified diet. In terms of supporting maximum bone calcification, phosphorus absorption was marginal in two of the groups on the ‘natural ingredient’ diets. Because of their variable fluoride concentrations, and ill-defined compositions, the use of ‘natural ingredient’ diets in research should be avoided. Key words: intake, absorption, faecal excretion, urinary excretion, balance, retention, bone fluoride, enamel fluoride, plasma fluoride. dental fluorosis, bone mineralization, enamel mineralization, osteoporosis, e:AMP.
INTRODUCTION Much of our knowledge of the metabolic and toxicological characteristics of fluoride stems from studies with laboratory animals. Such studies usually employ
either a ‘natural ingredient’ diet or a well-defined, semipurified diet. There are several reasons to expect that the quantitative features of fluoride metabolism would be affected differently by these two diets. The ‘natural ingredient’ diets contain higher concentrations of calcium, phosphorus and fluoride, mainly in the form of bone meal. Because of the insolubility of bone meal, the bioavailability of these ions is more limited than that from semipurified diets, which typically contain dibasic calcium phosphate. For reasons that are not clear, however, there are reports that the reduction in bioavailability of fluoride from bone meal is highly variable. For example, based on the proportionality of bone fluoride levels among several groups of rats, Taylor et al. (196 1) estimated that only IO-15% of the fluoride in a diet containing bone meal was bioavailable. Ham and Smith (1954) Abbreviations:
AAS, atomic absorption spectrometry; ANOVA, analysis, of variance; HMDS, hexamethyldisiloxane; TISAB, total ionic strength adjustment buffers.
found absorption to average 76% of the amount ingested when a ‘normal’ diet was consumed. When a cereal containing bone meal (Pablum) was added to the diet, fluoride absorption averaged 54% but it ranged from 19 to 76% among the subjects. Trautner and Siebert (1986) reported that the bioavailability of fluoride given as bone meal tablets was only 6% of that from sodium fluoride. Trautner and Einwag (1987) found that the bioavailability of fluoride from bone meal tablets or calcium fluoride tablets was less than 20% of that from sodium fluoride tablets. Moreover, the ‘natural ingredient’ diets have lower caloric densities and contain higher levels of fibre, sodium, potassium and chloride. These factors would be expected to increase fluoride intake with food because of caloric requirements and with water because of the greater loss of water in the urine and faeces. The published record appears to contain no data with which to evaluate this hypothesis. To the extent that these effects may occur, they would offset the decrease in bioavailability associated with the ingestion of bone meal. The unexplained discrepancies concerning the bioavailability of fluoride from bone meal and the lack of knowledge about the effects of the variables mentioned in the preceding paragraph prompted 291
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my study. The information obtained should be particularly useful in comparing the results of otherwise similar studies that differed in the type of diet used, and in selecting the appropriate diet for studies of the effects of low levels of fluoride intake, effects such as dental fluorosis and tissue concentrations of CAMP. Further, low levels of calcium or phosphorus absorption, such as might occur from some ‘natural ingredient’ diets, can adversely affect the mineralization of enamel (Massler and Schour, 1952; Nanda ef al., 1974) and bone (Bernhart, Savini and Tomerelli, 1969). For these reasons, the purposes of my 6-week study with weanling rats were to determine the relative effects of ‘natural ingredient’ and semipurified diets on: (1) food and water intake; (2) faecal and urine output; and (3) the intake, absorption, excretion, balance, plasma and calcified tissue concentrations of fluoride, calcium and phosphorus. MATERIALS AND METHODS
Weanling female Sprague-Dawley rats (n = 50) were placed in pairs in plastic metabolism cages and allowed free access to low-fluoride food (0.76 parts/lo”; American Institute of Nutirition semipurified diet (AIN-76A), U.S. Biochemical Corp.) and deionized water. The next day the rats were weighed and randomly assigned to five groups of equal size. The groups were distinguished by the powdered diets that they received. Groups A, B and C were provided with ‘natural ingredient’ diets while Groups D and E continued to receive the semipurified diet. The ‘natural ingredient’ diets contained a variety of unrefined components, such as corn and wheat flakes, ground corn and oats, alfalfa meal, soybean meal, dried whey and bone meal, that were not contained in the semipurified diet. The semipurified diet had a sodium concentration of 0.11% while the diets of Groups A, B and C had concentrations of 0.31, 0.40 and 0.52%, respectively. The chloride levels were 30-40% higher than the sodium levels in every diet. The potassium concentrations of the diets were 0.35, 0.99, 1.10 and 0.87%, respectively. In the ‘natural ingredient’ diets, bone meal supplied 75-85% of the calcium and 60-70% of the phosphorus, whereas in the semipurified diet these ions were added as dibasic calcium phosphate. Table 1 shows the calcium, phosphorus and fluoride concentrations of the diets, which were determined as described below. The fluoride in the diets was not added deliberately Table
I. Calcium,
phosphorus and fluoride of the rat diets
concentrations
Group
Calcium (g/kg) Phosphorus (n/kg) Fluoiide (mgjkg)
A
B
C
12.9 7.9 27.5
II.6 6.7 Il.6
17.8 Il.3 45.2
D and
E
8.5 7.0 0.76
The groups were fed the following diets: A-Wayne rodent blox” 8604-00; B-Purina rodent laboratory chow” 5001; C-Teklad 6% rat/mouse diet”; D and E-US. Biochemical AIN-76A semipurified rat-mouse diet”.
but was a contaminant of bone meal (Groups A, B and C) and the mineral mixtures. Except for the first day of the 6-week study, when no additional fluoride was given to the rats of Groups D and E, Group D animals received their non-dietary fluoride by intraperitoneal injection (yday, 7 days/week) while the Group E animals received most of their fluoride via the drinking water. The amounts of fluoride injected or added to the drinking water were calculated to approximate the average daily fluoride intake by the rats of Groups A, B and C. The 24-h food and water intakes were determined gravimetrically twice each week and the fluoride doses for Groups D and E were updated accordingly. Twenty-four hour urine and faecal output determinations were made twice each week. To make the comparison of plasma fluoride levels more meaningful, all rats were given the low-fluoride diet and deionized water during the last 16 h of the study and the rats of Group D were not dosed on the day of death. These precautions were taken to avoid the high plasma fluoride values that might have been caused by recent injections, water intake or food intake. Finally, the rats were anaesthetized with diethyl ether and a blood sample was collected from the aorta. The rats were then killed and the distal epiphyses of the femurs and the mandibular incisors were removed and cleaned of soft tissues. Enamel from 1.5-2.0 mm on either side of the transitional zone was removed using a scapel blade. The enamel and bone samples were dried to constant weight at 100 + 5°C. The bones were ashed overnight in porcelain crucibles at 550 _t 10°C. The ashed bone samples were crushed into a fine powder. Duplicate portions of the individual ashed bone and dried enamel samples were analysed for fluoride and phosphorus. All analyses were done in duplicate. When the results did not agree within IO%, another analysis was done and the outlier was discarded. Using the ion-specific electrode, the fluoride levels of urine and water were determined after adding an equal volume of TISAB (Orion Research). The 24-h faecal samples from each cage were homogenized (Brinkmann Polytron) in known volumes of deionized water. Fluoride in food, faeces, plasma, bone and enamel was analysed after overnight diffusion using the HMDSfacilitated diffusion method of Taves (1968) as modified by Whitford (1989). This method involves the digestion of the sample in HMDS-saturated 1.5 N sulphuric acid. Portions of the acid digest were analysed for calcium and/or phosphorus. Calcium was determined by AAS and phosphorus by the method of Chen, Toribara and Warner (1956). Urine samples were analysed for sodium and potassium by flame photometry and for chloride by electrometric titration. The data for 24-h net absorption and balance were calculated as follows. Net absorption (hereafter called absorption) was calculated by subtracting the quantity excreted in the faeces from the quantity ingested each day. At least for calcium, this method tends to underestimate true absorption because of the secretion of endogenous calcium into the intestinal tract (Hansard and Crowder, 1957) but this was not considered important for the purposes of this study.
293
Fluoride, calcium and phosphorus metabolism Table ;!. Body weights and plasma concentrations
of guoride, calcium and phosphorus Group D
E
58.7 &-4.2”
71.7 f 3.1”
188.7+ 5.p
183.1+ 4.58
186.5+_4.2”
60.4f4.1a
74.1 +_3.1”
Initial body weight Final body weight Plasma (fluoride) Plasma (calcium) Plasma (phosphorus)
C 70.5 f 5.P
B
A
177.3* 5.58
173.0* 4.40 0.81 + 0.04a 10.8 i. O.la,b 7.5 &0.3&b
1.21+0.11* 10.3 f 0.2b 7.3 * 0.2a.b
0.82 & 0.03s 11.2 f 0.28 8.2 f 0.3”
2.17+OJXb 10 6 -t 0.2@ 6:9 f 0.3a.b
3.21 + 0.24” 10.7 + 0.1a.b 6.5 * 0.4b
Units: Body weight, 8, fluoride, pmol/l; calcium and phosphorus, mg%. Values in the same row sharing a common superscript letter are not significantly different. Table 3 shows the fluoride and phosphorus con~ntrations in calcified tissue. Groups D and E had significantly higher fluoride levels than Groups A, B and C; Group E was higher than Group D for both enamel and bone. When the comparison was restricted to the groups on a ‘natural ingredient’ diet, C > A > B for both enamel and bone. The fluoride levels in enamel were less than 10% of those in epiphyses in each group. The phosphorus concentrations among the groups were statistically identical. Table 4 shows the data for food and water intake, for faecal and urine output, and for fluoride intake, balance and related factors. The intake of food and water was higher in Groups A, B and C than in Groups D and E by overall averages of 15 and 56%, respectively. The quantities of faeces and urine excreted also were higher in the groups given ‘natural ingredient’ diets. The average urinary excretion rates of sodium, potassium and chloride by Groups D and
Fractional absorption was calculated by dividing the amount absorbed by the amount ingested and balance by subtracting the total amount excreted from the total intake inclmiing, in the case of Group D, the amount of fluoride injected. Fractional retention was calculated by dividing the balance by the total dose. The data are expressed as mean + SE. Statistical analyses were done using ANOVA and p < 0.05 was selected as the indic~Itor for significance. The Scheffe F-test was used as the post hoc test. RESULTS
Table 2 shows the body weights and the terminal plasma levels of fluoride, calcium and phosphorus. The injection (D) and water (E) groups had a higher plasma fluoride than the groups on ‘natural ingredient’ diets; Group E was higher than Group D. All values for calcium and phosphorus were within the normal ranges (Ringler and Dabich, 1979). Table 3. Fluoride and phosphorus
~n~ntrations . of ashed femur distal epiphysis and dried incisor transrtronal zone enamel Group
Femur (fluoride) Enamel (fluoride) Femur ~hosph.orus) Enamel (phosphorus)
A
B
C
D
534 * 168 48.8 & 2.9” 18.1 * 0.078 13.9 f 0.28’
384 + 12” 34.1 -i_1.6” 18.3 & 0.12” 13.4 * 0.37%
618 f 17a 57.0 f 1.4” 18.3 & 0.18” 13.0 f 0.368
1695f31b 161.5 i 7.0b 18.2 f 0.09” 13.8 + 0.34”
E 2672 + 227.1 + 18.1 * 13.8 f
121’ 7.6’ O.OP 0.38”
Units: fluoride, mg/kg; phosphorus, g%. Values in the same row sharing a common superscript letter are not significantly different. Table 4. Food and water intake, faecal and urinary output and fluoride intake, absorption, excretion and balance data Group Food intake Water intake Faecal output Urine output Fluoride intake, food Fluoride intake, food + water Fluoride intake, total Fluoride excretion, faecal Fluoride absorption, dietary Fractional fluoride abs, dietary Fluoride excretion, urinary Fluoride excretion, total Fluoride balance Fractional fluoride retention
A
B
C
D
E
26.4 k 0.Y.’ 51.9 * 1.2” 5.2 rf:0.2” 17.1 *o.!Y 724 i 12” 724 + 12” 724+ 12” 399 + 15” 321+ 17” 44.7 * 2.0” 38+2” 437 f 15” 287 + 17” 39.2 f 2.wb
28.5 + 0.7” 58 1 + 1 5’.b 6:4 : 0:2b 18.6 f 0.9” 333 F 8b 333 f 8b 333 + gb 179 * 7b 152+9b 45.5 * 2.1” 26 f 2” 205 f gb 126 r Sb 37.6 f 2.1’
30.9 f 0.6b 63.3 + 1.8b 7.2 * 0.2c 25.6 & 1.3b 1393 f 29c 1393 ;t 29’ 1393 i: 29’ 701 * 31C 690 k 37’ 48.9 & 2.2” 34+2” 135 f 32’ 659 * 37’ 46.6 i 2.2b
25.3 2 0.4’ 38.0 & 1.3c l.61:0.1d 9.8 t 0.9c 191 id 19 I: id 925 & I@ 28f4d -8k4d -41.2 k 21.0b 33 5 5” 60 + 7d 864+9d 93.6 & 0.7E
24.4 + 0.5” 36.1 + l.lc 1.5 $O.ld 1.5 + 0.6c 18 i: Id 910 + 1c 910 & 33d 96 & 8d 813 & 31” 89.3 + 0.8’ 56 + lb 152+ Ilb 756 + 3T 82.6 + l.2d
Food and water intake and faecal and urinary output expressed as g/24 h/cage (n = 60). All other data expressed as pg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Negative values indicate net secretion into the gastro-intestinal tract. Values in the same row sharing a common superscript letter :are not significantly different.
WHITFORD
G. M.
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Table 5. Calcium intake, absorption, excretion and balance data Group
Calcium intake Calcium excretion, faecal Calcium absorption Fractional calcium absorption Calcium excretion urinary Calcium excretion total Calcium balance Fractional calcium retention
A
B
339 f 6” 197 k 8” 141 + 8” 41.6+2.1” 3.5 + 0.3” 201+ 8” 135 + 8” 40.2 & 2.1”
340 * 13” 184k8” 154 + 12a.d 44.6 + 2.0” 5.5 & 0.4b 192k8” 150 & 12a.d 43.2 & 2.0”
C 552 + 320 + 234 + 41.3 f 3.3 * 324 + 231& 40.7 +
D 12b 13b 15b 2.4” 0.2” 12b 15b 2.4”
215+3’ 30+2’ 186 + 3c.d 86.4 & l.Ob 2.0 f 0.2’ 32 + 2c 184 + 3c.d 85.5 2 l.Ob
E 207 f 28 + 179 + 86.3 z 1.7 + 30 + 177 + 85.5:
4’ 2’ 4”.’ I.Ob 0.2’ 2’ 4C.d l.Ob
All data (mean + SE, n = 60) expressed as mg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Values in the same row sharing a common superscript
letter are not significantly different. E were 788, 1644 and 817pmol/day/cage, respectively (not shown). The excretion rates by Groups A, B and C were all significantly higher and averaged 2758, 5005 and 2906 pmol/day/cage, respectively. The faeces of the groups on the ‘natural ingredient’ diets were 52.8% water by weight and 21.3% of the dried material was ash (not shown). The faeces of the groups given semipurified diet had slightly less water (48.9%) and slightly more ash (22.7%). As intended, the average total daily intake of fluoride by Groups D and E (918 pg/day/cage) was close to the average of the other three groups (819 pg/day/cage). In Groups A, B and C, a nearly constant fraction (SO-55%) of the total daily intake was excreted with the faeces. In Group E only 11% of the total daily fluoride intake was excreted with the faeces; in Group D (injected group), 47% more fluoride was excreted with the faeces than was ingested with food. The fractional absorption of fluoride ranged from 44.7 to 48.9% among the groups on the ‘natural ingredient’ diets. It was 89.3% for Group E and -41.2% in Group D. The balances and fractional retentions of fluoride were highest in the groups given the semipurified diet. Tables 5 and 6 show the data for calcium and phosphorus intake and balance. The ratios of the quantities of these ions excreted in the faeces to the quantities ingested fell within a narrow range and were high (49-58%) in the groups on ‘natural ingredient’ diets, while in Groups D and E the ratios were relatively low (14%). The values for fractional absorption and fractional retention of these ions were similar among Groups A, B and C, but in Groups D
and E the values were significantly higher by a factor of approximately two. DISCUSSION
My results indicate that, compared with the AIN-76A semipurified diet, the consumption of a ‘natural ingredient’ diet leads to the ingestion of more food and water and the excretion of more faeces and urine. These findings were undoubtedly related to the compositions of the diets. The crude fibre content of the semipurified diet was 3.8% and it was the only fibre in that food. The crude fibre content of the ‘natural ingredient’ diets ranged from 4.3 to 5.8%. These diets also contained additional but unknown amounts of fibre that were associated with the various cereal components. Fibre provides little nutritive value, is largely unabsorbed and makes necessary the faecal excretion of additional water. In large part, this explains why Groups A, B and C ate more food and excreted more faeces. The additional water excreted with the faeces in these groups, 4.7 ml/day/cage, can be calculated from the ‘faecal output’ data in Table 4 and the fact that the water content of the faeces was about 50% in all groups. While this helps to explain part of the difference in water intake, it does not explain the 12 ml/day/cage difference in urine output. This difference can be attributed to the fact that the sodium, potassium and chloride concentrations of the ‘natural ingredient’ diets were 3-5 times higher than that of the semipurified diet. Most of the additional intake of these ions was eliminated via the urine which, in turn, resulted in the urinary
Table 6. Phosphorus intake, absorption, excretion and balance data Group Phosohorus intake Phosphorus excretion, faecal Phosphorus absorption Fractional phosphorus absorption Phosphorus excretion urinary Phosphorus excretion total Phosphorus balance Fractional phosphorus retention
A
B
C
D
E
207 + 4* 119&5” 88 & 5” 42.6 + 2.3” 19 4 + 0.7;’ 138;5” 69 k 5” 33.1 + 2.3”
191 + 4”.d 109 &-5” 81 +4” 43.0 f 2.3” 7.4 * 0.7s 117*5* 74 _+4” 39.2 5 2.4”,c
350 + 8’ 170 + 8” 180k9b 51.1 * 2.2b 29.2 i 1.3’ 199 + 8b 151 * 9s 42.6 k 2.2b,c
177 _+ 3C.d 23 + 2’ 154+3c 87. I f 0.Y 28.5 k 1.6’ 51 k3’ 125 f 3’ 71.2 f 1.5d
170*3c 22* 2c 148 k 3’ 87.3 _+0.9’ 30.3 + 1.7’ 52 k 3’ 118_+4’ 49.4 * 1.5d
All data (mean * SE, n = 60) expressed as mg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Values in the same row sharing a common superscript letter are not significantly different.
Fluoride, calcium and phosphorus metabolism
excretion of an osmotically equivalent amount of water and the ingestion of a like amount of water. The effects of dietary composition, especially fibre and salt concentrations, on food and water intake and the excretion of urine and faeces are rarely discussed and probably often overlooked, although, as my study shows, they may be profound. They also may be confounding as, for example, in studies of water fluoride concentrations sufficient to cause marginal enamel fluorosis in laboratory animals and humans. For example:, if the subjects of one study consumed a low-fibre and/or low-salt diet while the subjects of another consumed a high-fibre and/or high-salt diet, then fluorosis in the high-fibre/highsalt group would be more prevalent and more severe simply because more water-borne fluoride would have been ingested. It is likely that studies of the relationship between water fluoride levels and the prevalence of dental caries would also be affected by these variables. These considerations are not limited solely to fluoride but would apply to the effects of any water-borne substance. As outlined in the Introduction, there is ample evidence that the bioavailability of fluoride from bone meal is limited, albeit variable from study to study, and that certain cations reduce the absorption of fluoride. Fluoride intake ranged from 333 to 1393 pg/day/cage in my groups fed the ‘natural ingredient’ diets (Table 4). The faecal excretion of fluoride was directly proportional to fluoride intake so that the fractional absorption values of the three groups (45549%) were stati:stically identical. The homogeneity of these results allows a high level of confidence in the conclusion that approximately one-half of the fluoride in diets of this type is bioavailable in the growing rat. The marked variability in the data from earlier reports is difficult to explain but, at least for the studies done befcsre the introduction of the ionspecific electrode, analytical problems were almost certainly involved (V’enkateswarlu, 1990). The animals of Group E received most of their fluoride in the drink.ing water. Fractional fluoride absorption in this group was 89%, about twice that of the groups given the ‘natural ingredient’ diets. This value is close to or identical with that reported by several others fcmr the control groups in their studies with rats drinking water containing sodium fluoride (Weddle and Muhler, 1955), with rats fed a semipurified diet to which sodium fluoride had been added (Cerkleuski and Bills, 1985; Cerklewski and Ridlington, 1985; Cerklewski, 1986, 1987; Cerklewski, Ridlington and Bills, 1986) or with humans who drank fluoride-containing mineral water (Trautner and Siebcrt, 1986). It appears that the quantitative aspects of the gastro-intestinal handling of fluoride by rats and humans are very similar. The fluoride balances in Groups A, B and C were related closely to fluoride intake, which resulted in similar values for fractional retention that ranged from 38 to 47% (Table 4). These were similar to those reported by Lawrenz and Mitchell (1941) who also fed their rats a ‘natural ingredient’ diet. In Groups D and E, the balances of fluoride were higher than in the other groups and the fractional retention values were about twice as high. These marked differences were explained almost completely by the differences in
295
faecal fluoride excretion. As can be calculated from the data in Table 4, the retention of the fluoride that was absorbed (balance/amount absorbed) was much closer among the five groups-it ranged from 83% in Group B to 96% in Group C. It may be concluded, therefore, that the metabolism of absorbed fluoride was not affected markedly by the types of diets I used. The effects of the limited solubility of bone meal on the absorption of calcium and phosphorus---and perhaps of phytate on the absorption of calcium (Bronner and Harris, 1954)--tan be seen in Tables 5 and 6. To achieve maximum bone (and perhaps enamel and dentine) calcification in rats fed a semipurified diet, calcium and phosphorus intakes should be 60 and 50mg/day, respectively (National Research Council (NRC), 1978). Assuming an average fractional absorption of 85% (Tables 5 and 6), the corresponding ranges for absorbed calcium and phosphorus would be 5 1 and 43 mg/day, respectively. In the groups on ‘natural ingredient’ diets, therefore, calcium absorption was adequate but, in Groups A and B, phosphorus absorption was either equal to or slightly less than the 43 mg/day recommended by the NRC. This marginal deficiency may have been associated with less than maximum mineralization and growth of calcified tissue. Although the differences did not reach statistical significance, the final body weights were lowest in Groups A and B. These considerations suggest that some ‘natural ingredient’ diets may be inappropriate for studies of the quality and quantity of calcified tissues. My findings suggest that the concentrations and bioavailability of fluoride in some ‘natural ingredient’ diets could cause some degree of enamel fluorosis. Angmar-Mgnsson and Whitford (1984) found that weanling rats fed an AIN semipurified diet ([F] = 0.5 parts/106) and given fluoride via miniosmotic pumps at a rate of 140pg/day for 8 weeks had an average plasma fluoride of 1.5 pmol/l and an average fluoride level in dried femoral diaphyses of 352 parts/106, which would correspond to a level of about 580 parts/IO6 in the ash. Microradiographic analysis of the developing incisor enamel indicated that they were mildly fluorotic. In my study, the animals in Groups A, B and C absorbed from 75 to 345 gg/ day/rat. Their average plasma values ranged from 0.8 to 1.2 pmol/l and the fluoride concentrations in ashed femur epiphyses ranged from 384 to 618 parts/106. The plasma levels were undoubtedly higher during most of the study because the low-fluoride AIN diet was provided during the 16 h before the blood samples were collected. This conclusion is supported by the findings of Ekstrand ef al. (1981) that, among rats given 25 or 50 parts/lo6 fluoride in the drinking water for 9 weeks, the plasma fluoride levels declined by 25 and 39%, respectively, during a 12-h period of fasting. Therefore, it is likely that some degree of incisor enamel fluorosis was present, especially in Groups A and C. Clearly, the use of ‘natural ingredient’ diets should be discouraged in studies of dental fluorosis. It is also possible that tissue CAMP levels would be elevated in rats fed a ‘natural ingredient’ diet. Kleiner, Miller and Allmann (1979) found that the addition of 20 parts/lo6 fluoride to an otherwise low-fluoride diet resulted in significant elevations in
G. M. WHITFORD
296
tissue CAMP levels of rats. In other studies with rats, it was found that fluoride at a concentration of only 1 part/lo6 in the drinking water was associated with elevated CAMP levels (Allmann et al., 1978; Kleiner and Allmann, 1982). Although plasma fluoride levels were not determined in these studies, they certainly would have been lower than those that I observed. However, it has not always been found that lowlevel fluoride intake can cause increases in CAMP in tissue and body fluid (Ophaug, Wong and Singer, 1979; Edgar, Jenkins and Prudhoe, 1979; Ekstrand et al., 1986). Nevertheless, in such studies it would be prudent to select a semipurified diet with a lowfluoride concentration. Finally, I will mention two other, previously unreported, findings. First, the rats of Group D, which received nearly all of their fluoride by intraperitoneal injection, excreted 41% more fluoride in the faeces than was ingested with food. Apparently the levels of fluoride in plasma and interstitial fluid were higher than those in solution within the intestinal lumen for some time after each injection, which promoted diffusion in the secretory direction. This process probably occurred most extensively in the distal ileum and perhaps in the colon where, because of previous absorption from the stomach (Whitford and Pashley, 1984) and proximal small intestine, the concentrations of fluoride would be low. The net intestinal secretion of fluoride and the factors that determine its magnitude (for example, the concentration of unabsorbed calcium) require further study. The findings could have importance to the formulation of more accurate pharmacokinetic models of fluoride metabolism. The process might also be one of the factors responsible for the lack of a beneficial effect in some osteoporotic patients treated with fluoride, most of whom usually ingest additional doses of calcium (Riggs et al., 1982). Second, the fractional absorption of fluoride in Group E had a downward trend from 94 to 82% (p < 0.001) during the 6-week study. It is not known whether this trend continues throughout life but it, as well as the underlying mechanism, deserves further study for the same reasons as studies of the intestinal secretion of fluoride. Acknotl,/edgemenrs-The author gratefully acknowledges the expert technical assistance of Janet M. Augeri and Isis El Moslimany and expresses his appreciation to Dr J. G. Weatherred, Dr D. H. Pashley and N. L. Birdsong-Whitford for their critical evaluation of the manuscript. This work was supported by a grant from the Medical College of Georgia Research Institute and by Grant DE-061 13 from the National Institute of Dental Research. NIH, Bethesda, MD. REFERENCES Allmann
D. W., Miller
A. and Kleiner H. S. (1978) Effect of fluoridated water on 3’5’ cyclic AMP levels in various rat tissues. J. den/. Re.r. 57, f&l. Anemar-MBnsson B. and Whitford G. M. (1984) Enamel Riorosis related to plasma F levels in the rat. Curie.7 Res. 18, 25-32. Bernhart F. W.. Savini S. and Tomarelli R. M. (1969) Calcium and phosphorus requirements for maximal growth and mineralization of the rat. J. Nurr. 98, 443-448.
Bronner F. and Harris R. S. (1954) Studies in calcium metabolism. Effect of food phytates in calcium4s uptake in children on low-calcium breakfasts. J. Nurr. 54, 523-542. Cerklewski F. L. (1986) Enhancement of fluoride retention by low dietary chloride without manifestation of chloride dkficiency in ;he rat. J. Nufr. 116, 1752-1755. Cerklewski F. L. (1987)Influence of dietary magnesium on fluoride bioavailability in the rat. J. Nufr. 117, 496-500. Cerklewski F. L. and Bills N. (1985) Influence of dietary iodide on fluoride bioavailability in the rat. Nutr. Rep. Int. 32, 991-994. Cerklewski F. L. and Ridlington J. W. (1985) Influence of zinc and iron on dietary fluoride utilization in the rat. J. Nufr. 115, 1162-1167. Cerklewski F. L., Ridlington J. W. and Bills N. D. (1986) Influence of dietary chloride on fluoride bioavailability in the rat. J. Nurr. 116, 618-624. Chen P. S. Jr, Toribara T. Y. and Warner H. (1956) Microdetermination of phosphorus. Analyf. Chem. 28, 1756-1758. Edgar W. M., Jenkins G. N. and Prudhoe P. (1979) Urinary CAMP excretion in human subjects following single and divided doses of sodium fluoride. J. dent. Res. 58, 1229. Ekstrand J., Lange A., Ekberg 0. and Hammarstriim (1981) Relationship between plasma, dentin and bone fluoride concentrations in rats following long-term fluoride administration. Acla I’harmac. Toxicol. 48, 433-437. Ekstrand J., Waller R., Forsberg A. and Fredholm B. (1986) High doses of fluoride do not affect cyclic AMP levels in human and rat plasma or urine. &and. J. dent. Res. 94, 507-5 14. Ham M. P. and Smith M. D. (1954) Fluorine balance studies on three women. J. Nuir. 53, 225-232. Hansard S. L. and Crowder H. M. (1957) The physiological behavior of calcium in the rat. J. Nutr. 62, 325-339. Kleiner H. S. and Allmann D. W. (1982) The effects of fluoridated water on rat urine and tissue CAMP levels. Archs oral Biol. 27, 107-l 12. Kleiner H. S., Miller A. and Allmann D. W. (1979) Effect of dietarv fluoride on rat tissue 3’5’-cyclic AMP levels. J. dent. Res. 58, 1920. Lawrenz M. and Mitchell H. H. (1941) The effect of dietary calcium and phosphorus on the assimilation of dietary fluorine. J. Nutr. 22, 91-101. Massler M. and Schour 1. (1952) Relation of endemic dental fluorosis to malnutrition. J. Am. denr. Ass. 44, 156-165. Nanda R. S., Zipkin. I.. Doyle J. and Horowitz H. S. (1974) Factors affecting the prevalence of dental fluorosis in Lucknow, India. Archs oral Biol. 19, 781-792. National Research Council Committee on Animal Nutrition (1978) Nutrient requirements of laboratory animals. Nal. Res. Council, Not. Acad. Sci. Nat. Res. Council, Washington D.C. Ophaug R. H., Wong K. M. and Singer L. (1979) Lack of effect of fluoride on urinary CAMP excretion in rats. J. dent. Res. 58, 2036-2039. Riggs B. L., Seeman E., Hodgson S. F., Taves D. R. and O’Fallon W. M. (1982) Effect of the fluoride/ calcium regimen on vertebral fracture occurrence in postmenopausal osteoporosis. Near Engl. J. Med. 306, 446-450. Ringler D. H. and Dabich L. (1979) The Laboratory RUI, Volume 1. Biology and Diseases (Edited by Baker H. J.. Lindsey J. R. and Weisbroth S. H.) Chap. 5, pp. 105-121. Academic Press, New York. Taves D. R. (1968) Determination of submicromolar concentrations of fluoride in biological samples. Talunra 15, 1015S1023. Taylor J. M., Gardner D. E., Scott J. K., Maynard E. A., Downs W. L., Smith F. A. and Hodge H. C. (1961) Toxic effect of fluoride on the rat kidney. II. Chronic effects. Toxicol. Appl. Pharmuc. 3, 290-314.
Fluoride, calcium and phosphorus metabolism Trautner K. and Einwa8 J. (1987) Factors influencing the bioavailability of fluoride from calcium-rich, health-food products and CaF, in man. Archs oral Biol. 32,401-406. Trautner K. and Siebert (G. (1986) An experimental study of bio-availability of fluoride from dietary sources in man. Archs oral Biol. 31, 223-228. Venkateswarlu P. (1990) Evaluation of analytical methods for fluorine in biological and related materials. J. dent. Res. 69 (Special Issue), 514-521.
291
Weddle D. A. and Muhler J. C. (1955) Metabolism of small concentrations of fluorine in the albino rat. J. dent. Res. 34,900-909.
Whitford G. M. (1989) The Metabolism and Toxicity of Fluoride (Edited by Myers H. M.), Monographs in Oral Science 13, pp. 26-30. S. Karger, Base!. Whitford G. M. and Pashley D. H. (1984) Fluoride absorption: the influence of.gastric acidity. Cak. Tiss. Int. 36, 302-307.
0003-9969/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
FLUORI:DE, CALCIUM AND PHOSPHORUS METABOLISM IN THE RAT: COMPARISON OF ‘NATURAL IYNGREDIENT’ WITH SEMIPURIFIED DIETS G. M. WHITFORD Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, U.S.A. (Received 30 March 1990; accepted 14 November 1990)
Summary-Three groups of weanling female rats were fed different, commercially available, ‘natural ingredient’ diets containing 12,28 or 45 parts/lo6 F, mainly as bone meal, for six weeks. Two other groups were fed a low-fluoride (0.76 parts/106) semipurified diet. They received fluoride doses, either in the drinking water or by daily intraperitoneal injection, which were approximately equal to the average dose of the other three groups. Rats on the ‘natural ingredient’ diets ingested more food and water and excreted more faeces and urine, effects which were attributed to the higher amounts of dietary fibre, Na, K and Cl. Thus, at any given concentration of fluoride in the food or water, the level of fluoride ingestion and the ensuing efFects would be influenced by the type of diet used. The values for fractional fluoride absorption (45-49%) and retention (38-47%) were similar among the groups given ‘natural ingredient’ diets. In the groups given semipurified diet, the corresponding values were about twice as high with the exception that fractional absorption was negative (-41%) in the injected group, which indicated net intestinal secretion of fluoride. Fluoride balances and tissue concentrations were highest in the groups fed the semipurified diet, even though the level of intake was not always higher. The fractional values for calcium and phosphorus absorption (41-51%) and retention (33-43%) were also similar among the groups given ‘natural ingredient’ diets. The corresponding values were about twice as high in the groups fed the semipurified diet. In terms of supporting maximum bone calcification, phosphorus absorption was marginal in two of the groups on the ‘natural ingredient’ diets. Because of their variable fluoride concentrations, and ill-defined compositions, the use of ‘natural ingredient’ diets in research should be avoided. Key words: intake, absorption, faecal excretion, urinary excretion, balance, retention, bone fluoride, enamel fluoride, plasma fluoride. dental fluorosis, bone mineralization, enamel mineralization, osteoporosis, e:AMP.
INTRODUCTION Much of our knowledge of the metabolic and toxicological characteristics of fluoride stems from studies with laboratory animals. Such studies usually employ
either a ‘natural ingredient’ diet or a well-defined, semipurified diet. There are several reasons to expect that the quantitative features of fluoride metabolism would be affected differently by these two diets. The ‘natural ingredient’ diets contain higher concentrations of calcium, phosphorus and fluoride, mainly in the form of bone meal. Because of the insolubility of bone meal, the bioavailability of these ions is more limited than that from semipurified diets, which typically contain dibasic calcium phosphate. For reasons that are not clear, however, there are reports that the reduction in bioavailability of fluoride from bone meal is highly variable. For example, based on the proportionality of bone fluoride levels among several groups of rats, Taylor et al. (196 1) estimated that only IO-15% of the fluoride in a diet containing bone meal was bioavailable. Ham and Smith (1954) Abbreviations:
AAS, atomic absorption spectrometry; ANOVA, analysis, of variance; HMDS, hexamethyldisiloxane; TISAB, total ionic strength adjustment buffers.
found absorption to average 76% of the amount ingested when a ‘normal’ diet was consumed. When a cereal containing bone meal (Pablum) was added to the diet, fluoride absorption averaged 54% but it ranged from 19 to 76% among the subjects. Trautner and Siebert (1986) reported that the bioavailability of fluoride given as bone meal tablets was only 6% of that from sodium fluoride. Trautner and Einwag (1987) found that the bioavailability of fluoride from bone meal tablets or calcium fluoride tablets was less than 20% of that from sodium fluoride tablets. Moreover, the ‘natural ingredient’ diets have lower caloric densities and contain higher levels of fibre, sodium, potassium and chloride. These factors would be expected to increase fluoride intake with food because of caloric requirements and with water because of the greater loss of water in the urine and faeces. The published record appears to contain no data with which to evaluate this hypothesis. To the extent that these effects may occur, they would offset the decrease in bioavailability associated with the ingestion of bone meal. The unexplained discrepancies concerning the bioavailability of fluoride from bone meal and the lack of knowledge about the effects of the variables mentioned in the preceding paragraph prompted 291
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my study. The information obtained should be particularly useful in comparing the results of otherwise similar studies that differed in the type of diet used, and in selecting the appropriate diet for studies of the effects of low levels of fluoride intake, effects such as dental fluorosis and tissue concentrations of CAMP. Further, low levels of calcium or phosphorus absorption, such as might occur from some ‘natural ingredient’ diets, can adversely affect the mineralization of enamel (Massler and Schour, 1952; Nanda ef al., 1974) and bone (Bernhart, Savini and Tomerelli, 1969). For these reasons, the purposes of my 6-week study with weanling rats were to determine the relative effects of ‘natural ingredient’ and semipurified diets on: (1) food and water intake; (2) faecal and urine output; and (3) the intake, absorption, excretion, balance, plasma and calcified tissue concentrations of fluoride, calcium and phosphorus. MATERIALS AND METHODS
Weanling female Sprague-Dawley rats (n = 50) were placed in pairs in plastic metabolism cages and allowed free access to low-fluoride food (0.76 parts/lo”; American Institute of Nutirition semipurified diet (AIN-76A), U.S. Biochemical Corp.) and deionized water. The next day the rats were weighed and randomly assigned to five groups of equal size. The groups were distinguished by the powdered diets that they received. Groups A, B and C were provided with ‘natural ingredient’ diets while Groups D and E continued to receive the semipurified diet. The ‘natural ingredient’ diets contained a variety of unrefined components, such as corn and wheat flakes, ground corn and oats, alfalfa meal, soybean meal, dried whey and bone meal, that were not contained in the semipurified diet. The semipurified diet had a sodium concentration of 0.11% while the diets of Groups A, B and C had concentrations of 0.31, 0.40 and 0.52%, respectively. The chloride levels were 30-40% higher than the sodium levels in every diet. The potassium concentrations of the diets were 0.35, 0.99, 1.10 and 0.87%, respectively. In the ‘natural ingredient’ diets, bone meal supplied 75-85% of the calcium and 60-70% of the phosphorus, whereas in the semipurified diet these ions were added as dibasic calcium phosphate. Table 1 shows the calcium, phosphorus and fluoride concentrations of the diets, which were determined as described below. The fluoride in the diets was not added deliberately Table
I. Calcium,
phosphorus and fluoride of the rat diets
concentrations
Group
Calcium (g/kg) Phosphorus (n/kg) Fluoiide (mgjkg)
A
B
C
12.9 7.9 27.5
II.6 6.7 Il.6
17.8 Il.3 45.2
D and
E
8.5 7.0 0.76
The groups were fed the following diets: A-Wayne rodent blox” 8604-00; B-Purina rodent laboratory chow” 5001; C-Teklad 6% rat/mouse diet”; D and E-US. Biochemical AIN-76A semipurified rat-mouse diet”.
but was a contaminant of bone meal (Groups A, B and C) and the mineral mixtures. Except for the first day of the 6-week study, when no additional fluoride was given to the rats of Groups D and E, Group D animals received their non-dietary fluoride by intraperitoneal injection (yday, 7 days/week) while the Group E animals received most of their fluoride via the drinking water. The amounts of fluoride injected or added to the drinking water were calculated to approximate the average daily fluoride intake by the rats of Groups A, B and C. The 24-h food and water intakes were determined gravimetrically twice each week and the fluoride doses for Groups D and E were updated accordingly. Twenty-four hour urine and faecal output determinations were made twice each week. To make the comparison of plasma fluoride levels more meaningful, all rats were given the low-fluoride diet and deionized water during the last 16 h of the study and the rats of Group D were not dosed on the day of death. These precautions were taken to avoid the high plasma fluoride values that might have been caused by recent injections, water intake or food intake. Finally, the rats were anaesthetized with diethyl ether and a blood sample was collected from the aorta. The rats were then killed and the distal epiphyses of the femurs and the mandibular incisors were removed and cleaned of soft tissues. Enamel from 1.5-2.0 mm on either side of the transitional zone was removed using a scapel blade. The enamel and bone samples were dried to constant weight at 100 + 5°C. The bones were ashed overnight in porcelain crucibles at 550 _t 10°C. The ashed bone samples were crushed into a fine powder. Duplicate portions of the individual ashed bone and dried enamel samples were analysed for fluoride and phosphorus. All analyses were done in duplicate. When the results did not agree within IO%, another analysis was done and the outlier was discarded. Using the ion-specific electrode, the fluoride levels of urine and water were determined after adding an equal volume of TISAB (Orion Research). The 24-h faecal samples from each cage were homogenized (Brinkmann Polytron) in known volumes of deionized water. Fluoride in food, faeces, plasma, bone and enamel was analysed after overnight diffusion using the HMDSfacilitated diffusion method of Taves (1968) as modified by Whitford (1989). This method involves the digestion of the sample in HMDS-saturated 1.5 N sulphuric acid. Portions of the acid digest were analysed for calcium and/or phosphorus. Calcium was determined by AAS and phosphorus by the method of Chen, Toribara and Warner (1956). Urine samples were analysed for sodium and potassium by flame photometry and for chloride by electrometric titration. The data for 24-h net absorption and balance were calculated as follows. Net absorption (hereafter called absorption) was calculated by subtracting the quantity excreted in the faeces from the quantity ingested each day. At least for calcium, this method tends to underestimate true absorption because of the secretion of endogenous calcium into the intestinal tract (Hansard and Crowder, 1957) but this was not considered important for the purposes of this study.
293
Fluoride, calcium and phosphorus metabolism Table ;!. Body weights and plasma concentrations
of guoride, calcium and phosphorus Group D
E
58.7 &-4.2”
71.7 f 3.1”
188.7+ 5.p
183.1+ 4.58
186.5+_4.2”
60.4f4.1a
74.1 +_3.1”
Initial body weight Final body weight Plasma (fluoride) Plasma (calcium) Plasma (phosphorus)
C 70.5 f 5.P
B
A
177.3* 5.58
173.0* 4.40 0.81 + 0.04a 10.8 i. O.la,b 7.5 &0.3&b
1.21+0.11* 10.3 f 0.2b 7.3 * 0.2a.b
0.82 & 0.03s 11.2 f 0.28 8.2 f 0.3”
2.17+OJXb 10 6 -t 0.2@ 6:9 f 0.3a.b
3.21 + 0.24” 10.7 + 0.1a.b 6.5 * 0.4b
Units: Body weight, 8, fluoride, pmol/l; calcium and phosphorus, mg%. Values in the same row sharing a common superscript letter are not significantly different. Table 3 shows the fluoride and phosphorus con~ntrations in calcified tissue. Groups D and E had significantly higher fluoride levels than Groups A, B and C; Group E was higher than Group D for both enamel and bone. When the comparison was restricted to the groups on a ‘natural ingredient’ diet, C > A > B for both enamel and bone. The fluoride levels in enamel were less than 10% of those in epiphyses in each group. The phosphorus concentrations among the groups were statistically identical. Table 4 shows the data for food and water intake, for faecal and urine output, and for fluoride intake, balance and related factors. The intake of food and water was higher in Groups A, B and C than in Groups D and E by overall averages of 15 and 56%, respectively. The quantities of faeces and urine excreted also were higher in the groups given ‘natural ingredient’ diets. The average urinary excretion rates of sodium, potassium and chloride by Groups D and
Fractional absorption was calculated by dividing the amount absorbed by the amount ingested and balance by subtracting the total amount excreted from the total intake inclmiing, in the case of Group D, the amount of fluoride injected. Fractional retention was calculated by dividing the balance by the total dose. The data are expressed as mean + SE. Statistical analyses were done using ANOVA and p < 0.05 was selected as the indic~Itor for significance. The Scheffe F-test was used as the post hoc test. RESULTS
Table 2 shows the body weights and the terminal plasma levels of fluoride, calcium and phosphorus. The injection (D) and water (E) groups had a higher plasma fluoride than the groups on ‘natural ingredient’ diets; Group E was higher than Group D. All values for calcium and phosphorus were within the normal ranges (Ringler and Dabich, 1979). Table 3. Fluoride and phosphorus
~n~ntrations . of ashed femur distal epiphysis and dried incisor transrtronal zone enamel Group
Femur (fluoride) Enamel (fluoride) Femur ~hosph.orus) Enamel (phosphorus)
A
B
C
D
534 * 168 48.8 & 2.9” 18.1 * 0.078 13.9 f 0.28’
384 + 12” 34.1 -i_1.6” 18.3 & 0.12” 13.4 * 0.37%
618 f 17a 57.0 f 1.4” 18.3 & 0.18” 13.0 f 0.368
1695f31b 161.5 i 7.0b 18.2 f 0.09” 13.8 + 0.34”
E 2672 + 227.1 + 18.1 * 13.8 f
121’ 7.6’ O.OP 0.38”
Units: fluoride, mg/kg; phosphorus, g%. Values in the same row sharing a common superscript letter are not significantly different. Table 4. Food and water intake, faecal and urinary output and fluoride intake, absorption, excretion and balance data Group Food intake Water intake Faecal output Urine output Fluoride intake, food Fluoride intake, food + water Fluoride intake, total Fluoride excretion, faecal Fluoride absorption, dietary Fractional fluoride abs, dietary Fluoride excretion, urinary Fluoride excretion, total Fluoride balance Fractional fluoride retention
A
B
C
D
E
26.4 k 0.Y.’ 51.9 * 1.2” 5.2 rf:0.2” 17.1 *o.!Y 724 i 12” 724 + 12” 724+ 12” 399 + 15” 321+ 17” 44.7 * 2.0” 38+2” 437 f 15” 287 + 17” 39.2 f 2.wb
28.5 + 0.7” 58 1 + 1 5’.b 6:4 : 0:2b 18.6 f 0.9” 333 F 8b 333 f 8b 333 + gb 179 * 7b 152+9b 45.5 * 2.1” 26 f 2” 205 f gb 126 r Sb 37.6 f 2.1’
30.9 f 0.6b 63.3 + 1.8b 7.2 * 0.2c 25.6 & 1.3b 1393 f 29c 1393 ;t 29’ 1393 i: 29’ 701 * 31C 690 k 37’ 48.9 & 2.2” 34+2” 135 f 32’ 659 * 37’ 46.6 i 2.2b
25.3 2 0.4’ 38.0 & 1.3c l.61:0.1d 9.8 t 0.9c 191 id 19 I: id 925 & I@ 28f4d -8k4d -41.2 k 21.0b 33 5 5” 60 + 7d 864+9d 93.6 & 0.7E
24.4 + 0.5” 36.1 + l.lc 1.5 $O.ld 1.5 + 0.6c 18 i: Id 910 + 1c 910 & 33d 96 & 8d 813 & 31” 89.3 + 0.8’ 56 + lb 152+ Ilb 756 + 3T 82.6 + l.2d
Food and water intake and faecal and urinary output expressed as g/24 h/cage (n = 60). All other data expressed as pg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Negative values indicate net secretion into the gastro-intestinal tract. Values in the same row sharing a common superscript letter :are not significantly different.
WHITFORD
G. M.
294
Table 5. Calcium intake, absorption, excretion and balance data Group
Calcium intake Calcium excretion, faecal Calcium absorption Fractional calcium absorption Calcium excretion urinary Calcium excretion total Calcium balance Fractional calcium retention
A
B
339 f 6” 197 k 8” 141 + 8” 41.6+2.1” 3.5 + 0.3” 201+ 8” 135 + 8” 40.2 & 2.1”
340 * 13” 184k8” 154 + 12a.d 44.6 + 2.0” 5.5 & 0.4b 192k8” 150 & 12a.d 43.2 & 2.0”
C 552 + 320 + 234 + 41.3 f 3.3 * 324 + 231& 40.7 +
D 12b 13b 15b 2.4” 0.2” 12b 15b 2.4”
215+3’ 30+2’ 186 + 3c.d 86.4 & l.Ob 2.0 f 0.2’ 32 + 2c 184 + 3c.d 85.5 2 l.Ob
E 207 f 28 + 179 + 86.3 z 1.7 + 30 + 177 + 85.5:
4’ 2’ 4”.’ I.Ob 0.2’ 2’ 4C.d l.Ob
All data (mean + SE, n = 60) expressed as mg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Values in the same row sharing a common superscript
letter are not significantly different. E were 788, 1644 and 817pmol/day/cage, respectively (not shown). The excretion rates by Groups A, B and C were all significantly higher and averaged 2758, 5005 and 2906 pmol/day/cage, respectively. The faeces of the groups on the ‘natural ingredient’ diets were 52.8% water by weight and 21.3% of the dried material was ash (not shown). The faeces of the groups given semipurified diet had slightly less water (48.9%) and slightly more ash (22.7%). As intended, the average total daily intake of fluoride by Groups D and E (918 pg/day/cage) was close to the average of the other three groups (819 pg/day/cage). In Groups A, B and C, a nearly constant fraction (SO-55%) of the total daily intake was excreted with the faeces. In Group E only 11% of the total daily fluoride intake was excreted with the faeces; in Group D (injected group), 47% more fluoride was excreted with the faeces than was ingested with food. The fractional absorption of fluoride ranged from 44.7 to 48.9% among the groups on the ‘natural ingredient’ diets. It was 89.3% for Group E and -41.2% in Group D. The balances and fractional retentions of fluoride were highest in the groups given the semipurified diet. Tables 5 and 6 show the data for calcium and phosphorus intake and balance. The ratios of the quantities of these ions excreted in the faeces to the quantities ingested fell within a narrow range and were high (49-58%) in the groups on ‘natural ingredient’ diets, while in Groups D and E the ratios were relatively low (14%). The values for fractional absorption and fractional retention of these ions were similar among Groups A, B and C, but in Groups D
and E the values were significantly higher by a factor of approximately two. DISCUSSION
My results indicate that, compared with the AIN-76A semipurified diet, the consumption of a ‘natural ingredient’ diet leads to the ingestion of more food and water and the excretion of more faeces and urine. These findings were undoubtedly related to the compositions of the diets. The crude fibre content of the semipurified diet was 3.8% and it was the only fibre in that food. The crude fibre content of the ‘natural ingredient’ diets ranged from 4.3 to 5.8%. These diets also contained additional but unknown amounts of fibre that were associated with the various cereal components. Fibre provides little nutritive value, is largely unabsorbed and makes necessary the faecal excretion of additional water. In large part, this explains why Groups A, B and C ate more food and excreted more faeces. The additional water excreted with the faeces in these groups, 4.7 ml/day/cage, can be calculated from the ‘faecal output’ data in Table 4 and the fact that the water content of the faeces was about 50% in all groups. While this helps to explain part of the difference in water intake, it does not explain the 12 ml/day/cage difference in urine output. This difference can be attributed to the fact that the sodium, potassium and chloride concentrations of the ‘natural ingredient’ diets were 3-5 times higher than that of the semipurified diet. Most of the additional intake of these ions was eliminated via the urine which, in turn, resulted in the urinary
Table 6. Phosphorus intake, absorption, excretion and balance data Group Phosohorus intake Phosphorus excretion, faecal Phosphorus absorption Fractional phosphorus absorption Phosphorus excretion urinary Phosphorus excretion total Phosphorus balance Fractional phosphorus retention
A
B
C
D
E
207 + 4* 119&5” 88 & 5” 42.6 + 2.3” 19 4 + 0.7;’ 138;5” 69 k 5” 33.1 + 2.3”
191 + 4”.d 109 &-5” 81 +4” 43.0 f 2.3” 7.4 * 0.7s 117*5* 74 _+4” 39.2 5 2.4”,c
350 + 8’ 170 + 8” 180k9b 51.1 * 2.2b 29.2 i 1.3’ 199 + 8b 151 * 9s 42.6 k 2.2b,c
177 _+ 3C.d 23 + 2’ 154+3c 87. I f 0.Y 28.5 k 1.6’ 51 k3’ 125 f 3’ 71.2 f 1.5d
170*3c 22* 2c 148 k 3’ 87.3 _+0.9’ 30.3 + 1.7’ 52 k 3’ 118_+4’ 49.4 * 1.5d
All data (mean * SE, n = 60) expressed as mg/24 h/cage except for fractional excretion and fractional retention which are percentages. There were two rats in each cage. Values in the same row sharing a common superscript letter are not significantly different.
Fluoride, calcium and phosphorus metabolism
excretion of an osmotically equivalent amount of water and the ingestion of a like amount of water. The effects of dietary composition, especially fibre and salt concentrations, on food and water intake and the excretion of urine and faeces are rarely discussed and probably often overlooked, although, as my study shows, they may be profound. They also may be confounding as, for example, in studies of water fluoride concentrations sufficient to cause marginal enamel fluorosis in laboratory animals and humans. For example:, if the subjects of one study consumed a low-fibre and/or low-salt diet while the subjects of another consumed a high-fibre and/or high-salt diet, then fluorosis in the high-fibre/highsalt group would be more prevalent and more severe simply because more water-borne fluoride would have been ingested. It is likely that studies of the relationship between water fluoride levels and the prevalence of dental caries would also be affected by these variables. These considerations are not limited solely to fluoride but would apply to the effects of any water-borne substance. As outlined in the Introduction, there is ample evidence that the bioavailability of fluoride from bone meal is limited, albeit variable from study to study, and that certain cations reduce the absorption of fluoride. Fluoride intake ranged from 333 to 1393 pg/day/cage in my groups fed the ‘natural ingredient’ diets (Table 4). The faecal excretion of fluoride was directly proportional to fluoride intake so that the fractional absorption values of the three groups (45549%) were stati:stically identical. The homogeneity of these results allows a high level of confidence in the conclusion that approximately one-half of the fluoride in diets of this type is bioavailable in the growing rat. The marked variability in the data from earlier reports is difficult to explain but, at least for the studies done befcsre the introduction of the ionspecific electrode, analytical problems were almost certainly involved (V’enkateswarlu, 1990). The animals of Group E received most of their fluoride in the drink.ing water. Fractional fluoride absorption in this group was 89%, about twice that of the groups given the ‘natural ingredient’ diets. This value is close to or identical with that reported by several others fcmr the control groups in their studies with rats drinking water containing sodium fluoride (Weddle and Muhler, 1955), with rats fed a semipurified diet to which sodium fluoride had been added (Cerkleuski and Bills, 1985; Cerklewski and Ridlington, 1985; Cerklewski, 1986, 1987; Cerklewski, Ridlington and Bills, 1986) or with humans who drank fluoride-containing mineral water (Trautner and Siebcrt, 1986). It appears that the quantitative aspects of the gastro-intestinal handling of fluoride by rats and humans are very similar. The fluoride balances in Groups A, B and C were related closely to fluoride intake, which resulted in similar values for fractional retention that ranged from 38 to 47% (Table 4). These were similar to those reported by Lawrenz and Mitchell (1941) who also fed their rats a ‘natural ingredient’ diet. In Groups D and E, the balances of fluoride were higher than in the other groups and the fractional retention values were about twice as high. These marked differences were explained almost completely by the differences in
295
faecal fluoride excretion. As can be calculated from the data in Table 4, the retention of the fluoride that was absorbed (balance/amount absorbed) was much closer among the five groups-it ranged from 83% in Group B to 96% in Group C. It may be concluded, therefore, that the metabolism of absorbed fluoride was not affected markedly by the types of diets I used. The effects of the limited solubility of bone meal on the absorption of calcium and phosphorus---and perhaps of phytate on the absorption of calcium (Bronner and Harris, 1954)--tan be seen in Tables 5 and 6. To achieve maximum bone (and perhaps enamel and dentine) calcification in rats fed a semipurified diet, calcium and phosphorus intakes should be 60 and 50mg/day, respectively (National Research Council (NRC), 1978). Assuming an average fractional absorption of 85% (Tables 5 and 6), the corresponding ranges for absorbed calcium and phosphorus would be 5 1 and 43 mg/day, respectively. In the groups on ‘natural ingredient’ diets, therefore, calcium absorption was adequate but, in Groups A and B, phosphorus absorption was either equal to or slightly less than the 43 mg/day recommended by the NRC. This marginal deficiency may have been associated with less than maximum mineralization and growth of calcified tissue. Although the differences did not reach statistical significance, the final body weights were lowest in Groups A and B. These considerations suggest that some ‘natural ingredient’ diets may be inappropriate for studies of the quality and quantity of calcified tissues. My findings suggest that the concentrations and bioavailability of fluoride in some ‘natural ingredient’ diets could cause some degree of enamel fluorosis. Angmar-Mgnsson and Whitford (1984) found that weanling rats fed an AIN semipurified diet ([F] = 0.5 parts/106) and given fluoride via miniosmotic pumps at a rate of 140pg/day for 8 weeks had an average plasma fluoride of 1.5 pmol/l and an average fluoride level in dried femoral diaphyses of 352 parts/106, which would correspond to a level of about 580 parts/IO6 in the ash. Microradiographic analysis of the developing incisor enamel indicated that they were mildly fluorotic. In my study, the animals in Groups A, B and C absorbed from 75 to 345 gg/ day/rat. Their average plasma values ranged from 0.8 to 1.2 pmol/l and the fluoride concentrations in ashed femur epiphyses ranged from 384 to 618 parts/106. The plasma levels were undoubtedly higher during most of the study because the low-fluoride AIN diet was provided during the 16 h before the blood samples were collected. This conclusion is supported by the findings of Ekstrand ef al. (1981) that, among rats given 25 or 50 parts/lo6 fluoride in the drinking water for 9 weeks, the plasma fluoride levels declined by 25 and 39%, respectively, during a 12-h period of fasting. Therefore, it is likely that some degree of incisor enamel fluorosis was present, especially in Groups A and C. Clearly, the use of ‘natural ingredient’ diets should be discouraged in studies of dental fluorosis. It is also possible that tissue CAMP levels would be elevated in rats fed a ‘natural ingredient’ diet. Kleiner, Miller and Allmann (1979) found that the addition of 20 parts/lo6 fluoride to an otherwise low-fluoride diet resulted in significant elevations in
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tissue CAMP levels of rats. In other studies with rats, it was found that fluoride at a concentration of only 1 part/lo6 in the drinking water was associated with elevated CAMP levels (Allmann et al., 1978; Kleiner and Allmann, 1982). Although plasma fluoride levels were not determined in these studies, they certainly would have been lower than those that I observed. However, it has not always been found that lowlevel fluoride intake can cause increases in CAMP in tissue and body fluid (Ophaug, Wong and Singer, 1979; Edgar, Jenkins and Prudhoe, 1979; Ekstrand et al., 1986). Nevertheless, in such studies it would be prudent to select a semipurified diet with a lowfluoride concentration. Finally, I will mention two other, previously unreported, findings. First, the rats of Group D, which received nearly all of their fluoride by intraperitoneal injection, excreted 41% more fluoride in the faeces than was ingested with food. Apparently the levels of fluoride in plasma and interstitial fluid were higher than those in solution within the intestinal lumen for some time after each injection, which promoted diffusion in the secretory direction. This process probably occurred most extensively in the distal ileum and perhaps in the colon where, because of previous absorption from the stomach (Whitford and Pashley, 1984) and proximal small intestine, the concentrations of fluoride would be low. The net intestinal secretion of fluoride and the factors that determine its magnitude (for example, the concentration of unabsorbed calcium) require further study. The findings could have importance to the formulation of more accurate pharmacokinetic models of fluoride metabolism. The process might also be one of the factors responsible for the lack of a beneficial effect in some osteoporotic patients treated with fluoride, most of whom usually ingest additional doses of calcium (Riggs et al., 1982). Second, the fractional absorption of fluoride in Group E had a downward trend from 94 to 82% (p < 0.001) during the 6-week study. It is not known whether this trend continues throughout life but it, as well as the underlying mechanism, deserves further study for the same reasons as studies of the intestinal secretion of fluoride. Acknotl,/edgemenrs-The author gratefully acknowledges the expert technical assistance of Janet M. Augeri and Isis El Moslimany and expresses his appreciation to Dr J. G. Weatherred, Dr D. H. Pashley and N. L. Birdsong-Whitford for their critical evaluation of the manuscript. This work was supported by a grant from the Medical College of Georgia Research Institute and by Grant DE-061 13 from the National Institute of Dental Research. NIH, Bethesda, MD. REFERENCES Allmann
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A. and Kleiner H. S. (1978) Effect of fluoridated water on 3’5’ cyclic AMP levels in various rat tissues. J. den/. Re.r. 57, f&l. Anemar-MBnsson B. and Whitford G. M. (1984) Enamel Riorosis related to plasma F levels in the rat. Curie.7 Res. 18, 25-32. Bernhart F. W.. Savini S. and Tomarelli R. M. (1969) Calcium and phosphorus requirements for maximal growth and mineralization of the rat. J. Nurr. 98, 443-448.
Bronner F. and Harris R. S. (1954) Studies in calcium metabolism. Effect of food phytates in calcium4s uptake in children on low-calcium breakfasts. J. Nurr. 54, 523-542. Cerklewski F. L. (1986) Enhancement of fluoride retention by low dietary chloride without manifestation of chloride dkficiency in ;he rat. J. Nufr. 116, 1752-1755. Cerklewski F. L. (1987)Influence of dietary magnesium on fluoride bioavailability in the rat. J. Nufr. 117, 496-500. Cerklewski F. L. and Bills N. (1985) Influence of dietary iodide on fluoride bioavailability in the rat. Nutr. Rep. Int. 32, 991-994. Cerklewski F. L. and Ridlington J. W. (1985) Influence of zinc and iron on dietary fluoride utilization in the rat. J. Nufr. 115, 1162-1167. Cerklewski F. L., Ridlington J. W. and Bills N. D. (1986) Influence of dietary chloride on fluoride bioavailability in the rat. J. Nurr. 116, 618-624. Chen P. S. Jr, Toribara T. Y. and Warner H. (1956) Microdetermination of phosphorus. Analyf. Chem. 28, 1756-1758. Edgar W. M., Jenkins G. N. and Prudhoe P. (1979) Urinary CAMP excretion in human subjects following single and divided doses of sodium fluoride. J. dent. Res. 58, 1229. Ekstrand J., Lange A., Ekberg 0. and Hammarstriim (1981) Relationship between plasma, dentin and bone fluoride concentrations in rats following long-term fluoride administration. Acla I’harmac. Toxicol. 48, 433-437. Ekstrand J., Waller R., Forsberg A. and Fredholm B. (1986) High doses of fluoride do not affect cyclic AMP levels in human and rat plasma or urine. &and. J. dent. Res. 94, 507-5 14. Ham M. P. and Smith M. D. (1954) Fluorine balance studies on three women. J. Nuir. 53, 225-232. Hansard S. L. and Crowder H. M. (1957) The physiological behavior of calcium in the rat. J. Nutr. 62, 325-339. Kleiner H. S. and Allmann D. W. (1982) The effects of fluoridated water on rat urine and tissue CAMP levels. Archs oral Biol. 27, 107-l 12. Kleiner H. S., Miller A. and Allmann D. W. (1979) Effect of dietarv fluoride on rat tissue 3’5’-cyclic AMP levels. J. dent. Res. 58, 1920. Lawrenz M. and Mitchell H. H. (1941) The effect of dietary calcium and phosphorus on the assimilation of dietary fluorine. J. Nutr. 22, 91-101. Massler M. and Schour 1. (1952) Relation of endemic dental fluorosis to malnutrition. J. Am. denr. Ass. 44, 156-165. Nanda R. S., Zipkin. I.. Doyle J. and Horowitz H. S. (1974) Factors affecting the prevalence of dental fluorosis in Lucknow, India. Archs oral Biol. 19, 781-792. National Research Council Committee on Animal Nutrition (1978) Nutrient requirements of laboratory animals. Nal. Res. Council, Not. Acad. Sci. Nat. Res. Council, Washington D.C. Ophaug R. H., Wong K. M. and Singer L. (1979) Lack of effect of fluoride on urinary CAMP excretion in rats. J. dent. Res. 58, 2036-2039. Riggs B. L., Seeman E., Hodgson S. F., Taves D. R. and O’Fallon W. M. (1982) Effect of the fluoride/ calcium regimen on vertebral fracture occurrence in postmenopausal osteoporosis. Near Engl. J. Med. 306, 446-450. Ringler D. H. and Dabich L. (1979) The Laboratory RUI, Volume 1. Biology and Diseases (Edited by Baker H. J.. Lindsey J. R. and Weisbroth S. H.) Chap. 5, pp. 105-121. Academic Press, New York. Taves D. R. (1968) Determination of submicromolar concentrations of fluoride in biological samples. Talunra 15, 1015S1023. Taylor J. M., Gardner D. E., Scott J. K., Maynard E. A., Downs W. L., Smith F. A. and Hodge H. C. (1961) Toxic effect of fluoride on the rat kidney. II. Chronic effects. Toxicol. Appl. Pharmuc. 3, 290-314.
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Whitford G. M. (1989) The Metabolism and Toxicity of Fluoride (Edited by Myers H. M.), Monographs in Oral Science 13, pp. 26-30. S. Karger, Base!. Whitford G. M. and Pashley D. H. (1984) Fluoride absorption: the influence of.gastric acidity. Cak. Tiss. Int. 36, 302-307.
