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Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet
Fd Chem. Toxic. Vol. 27, No. 1, pp. 1-9, 1989 Printed in Great Britain. All rights reserved
0278-6915/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc
Research Section EFFECT OF S O D I U M S A C C H A R I N A N D C A L C I U M S A C C H A R I N ON U R I N A R Y P A R A M E T E R S IN RATS F E D PROLAB 3200 OR AIN-76 DIET M. J. FISHER, T. SAKATA,T. S. TIBBELS, R. A. SMITH, K. PATIL, M. KHACHAB, S. L. JOH^NSSON and S. M. COHEN* Department of Pathology & Microbiology and the Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68105, USA (Received 4 April 1988; revisions received 8 September 1988)
Abstract--The effects of the salt form of saccharin and of diet on urinary ion levels have been studied in rats. Sodium saccharin (NaS) or calcium saccharin (CaS) was fed at a level of 5% in either Agway Prolab 3200 diet or AIN-76 diet to male, 5-wk-old F344 rats for l0 wk. The AIN-76 diet contained considerably less calcium, sodium and potassium than the Prolab 3200 diet, and smaller amounts of these ions were eliminated over 24 hr in the urine of rats fed the AIN-76 diet. Although food consumption was less in the groups fed AIN-76, total urinary saccharinate ion excretion with either saccharin salt was comparable with, or even higher than, that excreted by rats fed either salt in the Prolab 3200 diet. Rats fed Prolab 3200 eliminated approximately equal amounts of saccharinate ion in the faeces and urine. Rats fed AIN-76 eliminated about 10-20 times as much saccharin in the urine as in the faeces. Total saccharin excretion (faecal and urinary) was not influenced by the salt form. Water intake and urine volume were lower in rats fed control AIN-76 diet in comparison with those fed Prolab 3200, and were increased above the control level in groups fed saccharin in the AIN-76 diet. Urine electrolyte levels and osmolality were lower in the groups fed AIN-76. In general, NaS administration in either diet resulted in increased urinary sodium compared with controls, and the pH was at, or above, the level of control rats. CaS resulted in increased urinary calcium and decreased pH. There were marked diurnal variations in the urinary excretion of the various electrolytes, pH, and urine volume over a 24-hr period in all rats. This diurnal variation was more pronounced in the rats fed the Prolab 3200 diet. These results indicate that NaS and CaS have marked effects on the excretion of urinary electrolytes, and that these effects are influenced by diet.
similar in rats fed any one of the salt forms at comparable levels in the diet. However, there were marked variations in urinary pH and sodium, potassium, calcium and other ions when the different salt forms were administered (Hasegawa and Cohen, 1986). In most of the studies in our laboratory we have used the Prolab 3200 diet (originally referred to as Charles River diet) produced by Agway Inc. (St Marys, OH, USA). In related studies on saccharin a variety of diets have been used, including Purina no. 5002 (Schoenig et al., 1985), Oriental M F (Fukushima et al., 1983) and Wayne (Lawson and Hertzog, 1981). There are differences between these diets in the levels of minerals and other nutrients and these may result in numerous differences in the levels of urinary ions excreted. The present study was designed to compare the effects in male rats of diet, as well as saccharin salt form, on urinary ion levels. Also, since the excretion of substances in the urine is dependent on levels ingested and rats generally eat at night, we considered it important to determine the variations in ion concentrations over a 24-hr period. For diet comparisons, we selected the Prolab 3200 diet and a modified AIN-76 diet. Prolab 3200 is a closed-formula diet that is cereal based, unrefined, and unpurified. AIN-76 diet was developed by the American Institute of Nutrition as a purified, openformula diet that can be formulated exactly as specified (Committee on Standards for Nutritional
INTRODUCTION Sodium saccharin (NaS) increases the proliferation rate of the urothelium of the male rat when it is administered at high doses in the diet (Cohen, 1985; Fukushima and Cohen, 1980). The salt form in which saccharin is administered plays a critical role in the response of the bladder epithelium to the compound (Anderson et al., 1988; Hasegawa and Cohen, 1986; J. Ashby, personal communication, 1988). In a short-term proliferation assay (Hasegawa and Cohen, 1986) the increase in labelling index in the bladder epithelium following potassium saccharin administration was smaller than the response to sodium saccharin. Calcium saccharin (CaS) and acid saccharin had no effect on the labelling index in the same assay. This difference in biological responsiveness to the different forms of saccharin was not due simply to differences in absorption from the gastro-intestinal tract or differences in excretion in the urine (Hasegawa and Cohen, 1986). Concentrations of the saccharinate anion in the urine were *To whom reprint requests should be addressed: Department of Pathology & Microbiology, University of Nebraska Medical Center, 42nd & Dewey Avenue, Omaha, NE 68105, USA. Abbreviations: CaS=calcium saccharin; HPLC=highperformance liquid chromatography; NaS=sodium saccharin. I FCT 2"}:I - - A
M. J. FlsrFr~ et al. Table I. Experimental design Group no.
Diet
1 2 3 4 5 6
Prolab 3200 Prolab 3200 Prolab 3200 AIN-76 AIN-76 AIN-76
Treatment Control Sodium saccharin (5%) Calcium saccharin (5%) Control Sodium saccharin (5%) Calcium saccharin (5*/,)
Studies, 1977 and 1980). There are significant differences between these two diets, including marked differences in the levels of specific minerals. The ash and mineral content of the AIN-76 diet is considerably less than that of the Prolab 3200 diet (Agway Inc., 1986; Committee on Standards for Nutritional Studies, 1977 and 1980). Semi-synthetic diets have been suggested for use in toxicology and carcinogenicity studies (discussed at the Workshop to Optimize Diet for Rodents in Chemical Carcinogenicity Studies, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 15-16 October, 1985), and therefore it is essential to study the effects of these diets on physiological parameters, such as mineral metabolism, that might influence the response to a test chemical. MATERIALS AND M E T H O D S
Weanling F344 rats (Charles River Breeding Laboratories, Inc. Kingston, NY, USA) were fed Prolab 3200 pelleted diet during a 1-wk quarantine period. At 5 wk of age, they were randomly divided into six groups of ten rats each, using the weightstratified method of Martin et al. (1984), and started on the test diets (Table 1). The rats were housed five to a cage in polycarbonate cages (16 x 18 x 20in.) with stainless-steel wire-bar covers (Lab Products, Inc., Maywood, N J, USA) and dry corn-cob bedding. They were kept at 24 + T~C and 50 + 20% relative humidity, with a 12-hr light/dark cycle. Food and tap-water were available ad lib. For each cage of rats, food and water consumption over 3-day periods were measured six times during the study. The chemical consumption for the 10wk of the experiment was estimated by extrapolation of the food-consumption measurements. Body weights were determined periodically. NaS (lot no. 053F-0655, Sigma Chemical Co., St Louis, MO, USA) or CaS (provided by Dr T. Lawson, originally synthesized by Sherwin-Williams Co., Cincinnati, OH, USA) was mixed at a level of 5% by weight into either Prolab 3200 (Agway, Inc.), or AIN-76 diet (Committee on Standards for Nutritional Studies, 1977 and 1980). In the AIN-76 diet, the addition of saccharin was compensated for by a comparable reduction in dextrin, and the diet was prepared without added antioxidant and pelleted at the Eppley Institute for Research on Cancer under the supervision of Dr Diane Birt. The saccharin salts were mixed into Prolab 3200, and the diets were pelleted by Dyets Inc. (Bethlehem, PA, USA). The diets were stored in a freezer (-20~C) until used. Prolab 3200 and AIN-76 diets containing NaS were analysed (Hazleton Laboratories America Inc., Madison, WI, USA) for pH, protein, fat, moisture, ash, crude fibre (AOAC, 1984), carbohydrates,
calories (USDA, 1961) vitamins, minerals (Dahlquist and Knoll, 1978) and specific ions (AOAC, 1984). The pH of freshly voided urine was determined at the start of the study and at the end of wk I, 2, 3, 4, 6, 8 and 10 using a microelectrode (M1-40 Microcombination pH probe, Microelectrodes Inc., Londonderry, NH, USA). For these measurements, rats were removed individually from their cage beginning at 07.00 hr, and a drop of urine was expressed from the bladder onto a glass petri dish. The microelectrode was immediately immersed in the drop and the pH was determined. During wk 4 of the experiment, three rats from each group were housed individually in stainless-steel metabolism cages that were equipped for separation of urine and faeces (Model no. Spec. LC-176, Hazleton Co., Aberdeen, MD, USA). Food and water were available ad lib. Urine was collected at 4-hr intervals over 24 hr. On days 60 and 70 urine was collected from three rats from each group kept in metabolism cages for 24 hr (10.30-10.30 hr) and 22 hr (08.304)6.30 hr), respectively. Urine was collected via a funnel connected to a 50-ml polypropylene test tube. For 4-hr collection periods, the test tubes were placed in ice-filled plastic containers to prevent fermentation of the urine, and then the urine sample s were refrigerated at 4~C until they were analysed (within 24hr). For longer collection periods, I ml toluene was added to each test tube as a preservative. Urine pH was determined using the microelectrode immediately after each collection period was completed. At the end of each collection period, the faeces were removed from the metabolism cages, weighed and frozen ( - 6 ° C ) . The volume of urine was measured and then the urine was centrifuged (2000 rpm, 850g for 5 min; Sorvall T6000, Beckman Instruments Inc., Brea, CA, USA). Urinary concentrations of sodium, potassium, calcium, creatinine and urea were determined using the Beckman Astra 4 or 8 (Beckman Instruments Inc). Sodium and potassium concentrations (Tietz et al., 1986) were evaluated by an ion-specific electrode method (valinomycin for potassium). Calcium concentration was measured by a colorimetric method using o-cresolphthalein complex ion (Fraser et al., 1986). Creatinine concentration was determined using picric acid (Rock et al., 1986). Urea concentration was determined via the change in conductivity of urease due to ammonium ion and bicarbonate ion (Rock et al., 1986). Urinary osmolality (mOsm/kg) was measured by freezing point depression (Rock et al., 1986; Advanced Digimatic Osmometer, Model 301I, Advanced Instruments, Inc., Needham Heights, MA, USA). Samples of diet, faeces and urine were analysed for saccharin using a modification of the highperformance liquid chromatographic (HPLC) procedure of Tan & Pan (1982). Samples of diet or faeces (approximately 0.5 g each) were dried overnight in a single-wall transite oven (Blue M Electric Co., Blue Island, IL, USA), prehydrated with 0.5ml 0.1M-NaOH, extracted three times with 2.5ml 0.1 M-NaOH, and centrifuged (10min, 10002000 rpm) between each extraction. The supernatants were combined in a 10-ml volumetric flask and diluted to volume with triple-distilled water. The
Urinary function in rats fed saccharin salts AIN-76 diet was heated in a boiling water-bath during each extraction with NaOH in order to precipitate solubilized proteins that would otherwise give a gelatinous solution. Urine samples (I.0ml) were placed in a 10-ml volumetric flask, and diluted to volume with 7.5 ml 0.1 M-NaOH and !.5 ml H20. A l-ml aliquot of a stock solution of 0.122 M-NaS was treated in the same manner as urine for use as an extracted standard. An aliquot (0.5 ml) of the diet or faeces extract, diluted urine sample, or standard was applied to a 3-ml disposable (Baker Bond) quaternary amine (SAX) column that had been conditioned by sequential washes with methanol, ammonium hydroxide (sp. gr, 0.9), water and 0.2 M-HCI. After application of the sample, the column was washed with two 1-ml aliquots of methanol followed by one 2-ml aliquot of water. The saccharin was eluated into a 5-ml volumetric flask with 2-ml aliquots of 0.2M-K2HPO, buffer (pH 8.8), and then diluted to volume with the same buffer. HPLC was performed on a Waters (Waters Associates Inc., Milford, MA, USA) system consisting of a model 510 pump, U6K injector (250/~l loop), temperature control module oven, and a model 481 spectrophotometer interfaced with a HewlettPackard (Avondale, PA, USA) 3390A integrator. An aliquot (100/~l) of the sample was analysed using a Waters Novapak Ci8 column (15 cm x 0.39 cm i.d.) eluted with methanol-acetic acid-water (166:4:830, by vol, pH 3.5) at a flow rate of I ml/min at 30°C with UV detection at 254 nm, 0.5 AUFs. Experimental data was evaluated statistically on an IBM 4381 computer using a Generalized Linear Model procedure from the Statistical Analysis
3
System software package (SAS Institute, Inc., Car),, NC). Differences in body weight, diet and water intake, excretion of urine and faeces, saccharin consumption and excretion, and urinary sodium, potassium and calcium, were analysed at wk 4, 8 and 10. Multiple group comparisons were made simultaneously using Duncan's method. Diurnal analysis pertains to the wk 4 data, based on measurements of six consecutive 4-hr collections. RESULTS
Analyses of diets Analyses confirmed that there are numerous differences in the composition of the Prolab 3200 and AIN-76 diets (Table 2), and that the mineral content of AIN-76 is much lower than that of Prolab 3200. The calorific values of the AIN-76 and Prolab3200 diets are 380 and 345 kcal/100 g, respectively (International Life Sciences InstituteNutrition Foundation, personal communication, 1986).
Food and water consumption Throughout the experiment, control rats fed AIN-76 (Group 4) consumed less feed than did the controls fed Prolab 3200 (Group 1) (Table 3). Despite the greater calorific value of the AIN diet (Agway Inc., 1986; Committee on Standards for Nutritional Studies, 1977 and 1980), control rats fed AIN-76 diet consumed fewer calories than did those fed Prolab 3200 (calculated values not shown). It should be noted that pelleted Prolab 3200 is a softer diet than pelleted AIN-76. Although spillage was not significant in any of the cages included in food consumption
Table 2. Analysisof Prolab3200 and AIN-76diets AIN-76 Parameter Prolab3200 meal purifieddiet pH 6.3 7.5 Protein (%) 22.8 18.3 Moisture (%) 10.6 4.9 Fat (%) 5.8 5.1 Ash (%) 6.8 2.5 Crude fibre (%) 3.7 4.1 Carbohydrates (%) 50.3 65.l Calories (kcal/100g) 345 380 Cystine(rag/g) 3.82 0.69 Methionine(mg/g) 3.73 7.83 Chloride (ppm) 5040 1790 Bromide (ppm) < 100 < 100 Phosphate (ppm) 2180 1530 Nitrate-nitrogen(ppm) < 100 < 100 Nitrate-nitrogen(ppm) < 200 < 200 Sulphate (ppm) 597 912 Ca (ppm) 12,363 4843 Mg (ppm) 2401 497 K (ppm) 9350 3880 P (ppm) 9776 4975 Na (ppm) 2841 1018 AI (ppm) 107.9 12.0 Ba (ppm) 8.41 0.15 Fe (ppm) 299 51.6 Sr (ppm) 15.8 2.08 B (ppm) 7.23 1.00 Cu (ppm) I 1.0 4.I Zn (ppm) 82.0 33.I Mn (ppm) 68.7 46.2 Cr (ppm) 0.88 0.66
4
M.J. Ftsnr~ et al.
Table 3. Diet and water intake,saccharinconsumption,and body weight of rats fed sodium saccharinor calciumsaccharin in Prolab3200 or AIN-76diet Daily intake (mean ± SEM) of: Croup no. 1 2 3 4 5 6 l 2 3 4 5 6
Diet Prolab 3200
AIN-76
Prolab 3200
AIN-76
Treatment
No. o f rats
Body weight (mean + S E M g)
Diet (g)
Water (ml)
Control NaS CaS Control NaS CaS
10 10 10 10 10 9*
225 214 207 222 199 199
+ ± ± ± + ±
Wk5 3 3° M 3 2~ 3b
19 24 30 16 16 16
_+ 0.4 ~ _+ 0.7 ~ ~ 2.4 ~ + 0.3 ± 0.9 _+ 0.2
30 33 33 22 32 42
+_ 0.6 ~ + 1.6 + 0.4 d ~_ 0.4 _+ 0.5 ~ ± 4.6 ~
Control NaS CaS Control NaS CaS
l0 10 10 l0 10 9*
276 261 260 273 247 246
Wk 8 "~-4 ± 4~ ___4 ~ _ 3 + 3~ ± 4b
18 20 23 17 15 15
_+ 0.1 ~ +_ 0.3 '~ _+ 0.1 ~ + 0.2 _+ 0.2 ± 0.5
28 34 33 22 28 28
_+ 0.4 +_ 1.5 ~ _+ 0.7 ~ + 2.7 ° ± 0.2 ~ + 0.5 b
Control NaS CaS Control NaS CaS
10 10 10 10 10 9*
298 282 279 295 266 266
+ 4 +. 4* ± 5* ± 3 ± 4b ± 4b
17 18 23 14 14 14
_+ 0.2 + 0.1 ~ ± 1.0~ + 0.2 a _+ 0.2 _+ 0.1
23 33 35 17 24 25
_ 0.3 + 1.0'~ ± 0.6 *a ± 0.2* _+ 0.5 h _+ 2.0 b
Total consumption o f saccharin/rat at wk 10 (g)
D
m
m
Wk I0 1 2 3 4 5 6
Prolab 3200
AIN-76
N a S = sodium saccharin *One rat died during the experiment. Within each parameter, values m a r k e d Significantly different from G r o u p I, Significantly different from G r o u p 4, Significantly different from G r o u p 5, Significantly different from G r o u p 6,
0 56 73 0 34 36
C a S = calcium saccharin
with superscripts differ significantly as follows: Ap = 0.05, ' P ~ 0.01, ° P = 0.001; bp = 0.001; cp = 0.001; dp = 0.001.
measurements, it is possible that slightly more of the Prolab 3200 fell from the feeders into the bedding. The possibility that consumption of Prolab 3200 has been overestimated due to spillage is borne out by the body-weight data (Table 3). Consumption of saccharin-containing AIN-76 diets (Groups 5 and 6) was similar to that of the control AIN-76 diet. In contrast, rats fed Prolab 3200 ate more CaS diet (Group 3) than NaS diet (Group 2), and both of these groups consumed more feed than the control group did. The amount of saccharin consumed during the experiment (Table 3) was greater for Groups 2 and 3 than it was for Groups 5 and 6 because of the greater consumption of the Prolab 3200 diet. More water was consumed by the control group fed Prolab 3200 compared with the control group fed AIN-76 (Table 3). With both diets, feeding either NaS or CaS increased water consumption compared with the corresponding control group. Water consumption by the saccharin-fed rats was significantly greater in the Prolab 3200 groups than in the AIN-76 groups only during wk 10. Weight gain The mean body weights of the control groups (1 and 4) did not differ significantly at wk 4, 8 or 10. Rats fed NaS or CaS in either diet gained less weight than the corresponding control group (Table 3). This decreased weight gain was more notable in rats fed the AIN-76 diet. Faecal elimination o f saccharin The amount of faeces (wet weight) voided daily gradually increased during the experiment in rats fed Prolab 3200 but not in those fed AIN-76 (Table 4).
The amount of faeces voided per day was similar in all three groups fed AIN-76. The rats fed Prolab with NaS voided approximately the same amount of faeces per day as the control group fed Prolab. However, in contrast the group fed CaS in Prolab 3200 voided significantly more faeces at all three time points in comparison with either the control or NaS groups (Table 4). Except at wk 4, the saccharin concentration was similar in the faeces of rats fed Prolab 3200 containing NaS or CaS (Table 4). In contrast, rats fed CaS in AIN-76 had higher saccharin concentrations in the faeces than did rats fed NaS in AIN-76, although the differences were significant only at wk 10. The faecal saccharin concentration was lower in both AIN-saccharin groups than in the corresponding Prolab-saccharin groups. Given these differences in concentration, along with differences in the amount of faecal material voided, the total amount of saccharin eliminated in the faeces was appreciably higher in rats fed the Prolab diet than in those fed the AIN diet (Table 4). Also, with both diets, the total amount of saccharin eliminated in the faeces was greater in the rats fed CaS than in those fed NaS; this increase was statistically significant only for the Prolab diet. Urinary excretion o f saccharin Urine volume was increased in rats fed the AIN-76 diet containing either form of saccharin (Table 4). This saccharin-associated increase in urine volume was not seen in Prolab-fed groups during the time the rats were housed in the metabolism cages. Urinary saccharin concentration was measured only at 8 and l0 wk (Table 4). For rats fed a particular basal diet, there appeared to be no differences
Urinary function in rats fed saccharin salts
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Urinary function in rats fed saccharin salts associated with saccharin salt form in either urinary saccharin concentration or in the amount of saccharin voided over 24 hr. Although the urinary concentration of saccharin in rats fed the AIN-76 diet was less than that in the rats in the corresponding groups fed Prolab 3200 (this observation was statistically significant for the CaS-fed groups at wk 8), the higher urine volume in the AIN-saccharin groups resulted in a significantly larger amount of saccharin being excreted in the urine over 24 hr by the AIN-76 groups. This contrasts with the comparatively low levels of saccharin eliminated via the faeces in the AIN-76 groups. As a result, the ratio of urinary saccharin to faecal saccharin was dramatically higher in rats fed AIN-76 diet containing saccharin compared with that in rats fed the corresponding Prolab diets (Table 4). Saccharin elimination over 24 hr by rats fed Prolab 3200 diet was almost equally divided between the urine and faeces, whereas in rats fed the AIN-76 diet, 10-20 times as much saccharin was excreted in the urine as in the faeces.
Urinary parameters--longitudinal comparisons Except for one rat which was eliminated from G r o u p 3 at the 8-wk collection, all of the total urinary creatinine levels were within normal limits. Because at 4 wk the 24-hr urine colletion was divided into six 4-hr periods, insufficient sample was available for urea analysis. No significant differences were observed between any of the groups in total urea excretion in the urine at 8 or 10 wk. However, the concentration of urea was signifiantly higher in rats fed CaS in the Prolab diet at 10 wk, but not at 8 wk. Because of the marked increase in urinary volume, the concentration of urea in the rats fed the A I N diet containing NaS or CaS was significantly less than in the control group at both time points. There were no significant differences in total urea or urea concentration between rats fed NaS or CaS in either diet. Concentrations of N a ' , K + and Ca-" in urine samples collected over 24 hr are shown in Table 5. Urinary concentrations of Na + and K ÷ were lower for the rats fed the AIN-76 diet than for the corre-
450 [--
•
225 I.-
/ - ~ ' x \ \
1// ,5 P Z _ - o . -
oil 0
•
\\
0400
"-.
~'\
~i
i
"%=--~---~
0800
1200
.... 1600
2000
Time of duy
Fig. 2. Urinary sodium concentrations. Urine was collected over six consecutive 4-hr intervals. The time of day at which each interval ended is indicated. - - A - - = Group 1 (control Prolab 3200); - - O - - = Group 2 (Prolab 3200 + sodium saccharin); - - I 1 - - = Group 3 (Prolab3200+calcium saccharin); - - /~ . . . . Group 4 (control AIN-76); ---© - = Group 5 (AIN-76+sodium saccharin); [] . . . . Group 6 (AIN-76 + calcium saccharin). sponding Prolab 3200 groups. Similarly, the total levels of Na + and K + excreted in the urine over 24 hr were generally less in rats fed AIN-76 diet. The urinary concentration of Ca 2~ was also generally lower in the rats fed AIN-76 diet. Feeding either NaS or CaS increased the urinary concentration and excretion of the relevant cation in both the AIN-76 and Prolab 3200 groups. In rats fed AIN-76 containing saccharin, the urinary K + concentration was significantly less than in those fed the control AIN-76 diet; this difference was related to the difference in urine volume and, therefore, there were no differences between the groups fed AIN-76 diet in the total amount of K + excreted in the urine. The pH of the urine of groups fed the AIN-76 diets was significantly less than that of the urine of any of the groups fed Prolab 3200 (Fig. 1). Within the three AIN-76 groups, there was little difference in the urinary pH at any of the time points. The urinary pH of rats fed CaS in Prolab 3200 was significantly lower than the urinary pH of rats fed NaS in the same diet at wk 1, 2, 4, 6 and 8, and significantly lower than the Prolab controls at wk 1, 4, 6 and 10. In general, the urinary pH of the rats fed Prolab 3200 containing
8.0
./=
3501 7.5
'~ 300
"l" 7.0 'i
.~
6.5 .~. i ,,,O~ "
6.0
~=8>__
J>- ~ > ~ S _ _ ~ _ ~
200
~
~ 150
~ "~
Iooi
5.5 5.0
0
I 7
I 14
I 21
I 28
I I 35 42 Doy
I 49
I 56
I 65
J 70
Fig. 1. pH of freshly voided urine samples collected from rats at 07.00 hr at the specified weeks of the experiment. - - A - - = Group l (control Prolab 3200); - - O - - = Group 2 (Prolab 3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . . Group 4 (control AIN-76); - - - © . . . . Group 5 (AIN-76 + sodium saccharin); - - - [ ] . . . . . Group 6 (AIN-76 + calcium saccharin).
0
0
I 0400
I 0800
I 1200
I 1600
i 2000
Time of day
Fig. 3. Urine potassium concentrations. The indicated times represent the end of a 4-hr collection period. - - A - = Group I (control Prolab 3200); - - O - - = Group 2 (Pro]ab3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - O . . . . Group 5 (AIN-76 + sodium saccharin); ---17 .... Group 6 (AIN-76 + calcium
saccharin).
M. J. FLSHr,R et al. 150
relatively constant between 20.00 and 08.00 hr, but it was decreased at 12.00 and 16.00hr. In general, the diurnal variation of the different cations in the urine was greater in the rats fed Prolab 3200 than in those fed AIN-76. However, with all of these parameters, there was significant variation • within groups at the different time points.
100
°f 0
0
DISCUSSION
I 0400
I 0800
I 1200
I '1600
I 2000
Time of roy
Fig. 4. Urine calcium concentrations. The indicted times represent the end of a 4-hr collection period. - - A - - = Group 1 (control Prolab3200); - - O - - = G r o u p 2 (Prolab 3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - O = Group 5 (AIN-76 + sodium saccharin); - - - [ 3 - - = Group 6 (AIN-76+ calcium saccharin). NaS was similar to the urinary pH of rats fed the control Prolab diet. Urine osmolality was similar among the three groups fed Prolab 3200, whereas the urine from rats fed the AIN-76 diet containing NaS or CaS had a lower osmolality than the urine from the controls. The osmolality of the urine from control AIN-76 rats was less than that of urine from rats fed the control Prolab diet. D i u r n a l variations
The diurnal variations within urinary pH and concentrations of Na ÷, K ÷ and Ca 2+ are shown in Figs 2-5. Urinary concentrations of Na ÷ and K + were generally higher at night than during the day, with the lowest values usually occurring at 16.00 or 20.00hr. Urinary Ca 2÷ remained at approximately the same level throughout the 24-hr period except for an increase in concentration at 16.00 hr. Urinary pH was measured on freshly voided samples, rather than on collected samples (Fig. 5). There was less diurnal variation for the rats fed the different AIN-76 diets than for those fed the corresponding Prolab 3200 diets. In the rats fed Prolab 3200, the urinary pH was 8.0 7.5
Z el
Z /Z.
7.0 6.5 6.0 5.5
5.C
I 0400
I oeoo Time
I 1200 of
I 1600
I 2000
doy
Fig. 5. pH freshly voided urine from rats not contained in metabolism cages. - - A - - = Group I (control Prolab 3200); - - 0 - - = Group 2 (Prolab 3200 + sodium saccharin); - - I I - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - © . . . . . . Group 5 (AIN-76 + sodium saccharin); ---I-l . . . . Group 6 (AIN-76 + calcium saccharin).
The present experiment has confirmed the previous finding that rats fed NaS or CaS excrete similar levels of saccharin in the urine (Hasegawa and Cohen, 1986; Patricia Kraft, personal communication 1984). However, despite this similarity, there appear to be marked differences in urinary pH and the excretion of various electrolytes in the urine following administration of these two saccharin salt forms. These differences have been more clearly defined in the present study, as has the influence of administering NaS or CaS in two very different diets. Since the AIN-76 diet has more calories per gram than does the Prolab 3200 diet (International Life Sciences Institute-Nutrition Foundation, personal communication 1986), rats would be expected to consume less of the AIN-76 diet than the Prolab diet. Also, with saccharin present in the diet at such high levels, the rats would be expected to eat slightly greater amounts of the saccharin-containing diets than the control diets in order to compensate for the lower number of calories per gram of diet. Essentially this was observed in the present experiment, although the consumption of Prolab 3200 was even greater than would have been expected. It is possible that spillage of the feed contributed to the higher values of food consumption of Prolab 3200, since the groups fed control AIN-76 and Prolab 3200 gained weight at similar rates. For both CaS and NaS total, the dose of saccharin, calculated from data on food consumption and diet analyses, was less for rats fed the AIN-76 diet than for those fed the Prolab 3200 diet. A marked increase in urine volume was seen following administration of NaS or CaS in the AIN-76 diet, but there was no such increase with the Prolab 3200 diet (Table 4). The urinary saccharin concentration was similar regardless of the salt form or diet in which the saccharin was administered. The total amount of saccharin voided over 24 hr was not affected by saccharin salt form, but values for the rats fed AIN-76 were higher than those for rats fed Prolab3200 due to the increase in urine volume. Marked differences in the faecal excretion of saccharin were observed, and these were consistent with the differences in saccharin intake (Table 4). Rats fed AIN-76 diet excreted smaller amounts of faeces with lower concentrations of saccharin than did rats fed the corresponding Prolab 3200 diets. These findings with the Prolab 3200 diet are similar to those previously reported in rats fed NaS in Purina diet (Schoenig and Anderson, 1985). The AIN-76 diet has a lower mineral content than the Prolab 3200 diet (Table 2), and the rats fed AIN-76 excreted urine containing smaller amounts of Na ~, K + and Ca :+ than did those fed Prolab 3200. Of particular note was the consistently lower urinary pH observed in rats fed the control and saccharin-
Urinary function in rats fed saccharin salts containing AIN-76 diets, in contrast to the approximately neutral to slightly alkaline pH of the urine of rats fed Prolab 3200. The urinary pH of Prolab 3200fed rats increased slightly following administration of NaS and decreased following CaS administration. As would be anticipated, urinary Na ~ was considerably elevated in rats fed NaS, whether in the Prolab 3200 diet or AIN-76 diet; similarly, urinary Ca `'+ was elevated in rats fed CaS in either of the diets. Numerous studies (deGroot et al., 1988; Fukushima et al., 1986; Harguindey, 1982; Kritchevsky, 1986; Renwick and Sims, 1983; Schoenig and Anderson, 1985) have indicated an influence of pH, levels of sodium and calcium, urine volume, and calorie intake on the biological response to a variety of chemicals. Thus, it is important to study the levels of various ions in the urine or target tissues of rats fed high levels of various compounds in the diet. It is also essential to note that the time of observation of the various parameters is particulary important because of the striking diurnal variation in the urinary ion excretion (Sims and Renwick, 1984). There is marked variation in the urinary excretion of the various electrolytes, and in pH and urinary volume, over a 24-hr period, as would be anticipated considering the eating and drinking habits of the rat. We also observed that measurement of urinary pH is more accurate if done at the same time(s) each day, and carried out immediately using a microelectrode, rather than on collected specimens. Collected specimens vary considerably in pH over time. The present studies indicate that, although the level of saccharinate ion in the urine is similar, there are marked differences in the excretion of various ions (Figs 1-3; Table 2) by rats fed NaS in comparison with those fed CaS. Also, administering these compounds in two very different diets, Prolab 3200 and the AIN-76, results in marked differences in the excretion of various ions in the urine, although the saccharinate excretion appears similar. Acknowledgements--We gratefully acknowledge the advice and comments of Drs Emily Garland, Diane Birt, Gerald Schoenig and Jeffrey Huth, the technical assistance of Margaret St. John and James Gulizia, and the assistance of Jan Leemkuil in the preparation of this manuscript. This work was supported in part by a grant from the International Life Sciences Institute Nutrition Foundation, and by USPHS grant no. CA36727 from the National Cancer Institute. REFERENCES
Agway Inc. (1986). Prolab formulas for precise feeding of rats, mice, and hamsters. Agway Inc., St Mary's, OH, USA. Anderson R. L., Lefever F. R. and Maurer J. K. (1988). The effect of various saccharin forms on gastrointestinal tract, urine and bladder of male rats. Fd Chem. Toxic. 26, 665. AOA,S (1984). Official Methods of Analysis. Edited by S. Williams. 14th Ed. Association of Official Analytical Chemists, Arlington, VA, USA.
Cohen S. M. (1985). Multi-stage carcinogenesis in the urinary bladder. Fd Chem. Toxic. 23, 521. Committee on Standards for Nutritional Studies (1977). Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 107, 1340. Committee on Standards for Nutritional Studies (1980). Second report of the Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 110, 1726. Dahlquist R. L. and Knoll J. W. (1978). Inductively coupled plasma-atomic emission spectrometry: analysis of biological materials and soils for major, trace, and ultra-trace elements. Appl. Spectrosc. 32, 1. deGroot A. P., Feron V. J. and Imnael H. R. (1988). Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/carcinogenicity testing. Fd Chem. Toxic. 26, 425. Fraser D., Jones G., Kooh S. W. and Radde I. C. (1986). Textbook of Clinical Chemistry. Ist Ed. p. 1317. W. B. Saunders Co., Philadelphia, PA. Fukushima S., Arai M., Nakanowatari J., Hibino T., Okuda M. and lto N. (1983). Differences in susceptibility to sodium saccharin among various strains of rats and other animal species. Gann 74, 8. Fukushima S. and Cohen S. M. (1980). Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res. 40, 734. Fukushima S., Shibata M-A., Shirai T., Tamano S. and Ito N. (1986). Roles of urinary sodium ion concentration and pH in promotion by ascorbic acid of urinary bladder carcinogenesis in rats. Cancer Res. 46, 1623. Harguindey S. (1982). Hydrogen ion dynamics and cancer: an appraisal. Med. Pediat. Oncol. 10, 217. Hasegawa R. and Cohen S. M. (1986). The effect of different salts of saccharin on the rat urinary bladder. Cancer Lett. 30, 261. Kritchevsky D. (1986). Diet, nutrition, and cancer: the role of fiber. Cancer, N.Y. 58, 1830. Lawson T. A. and Hertzog P. J. (1981). The failure of chronically administered saccharin to stimulate bladder epithelial DNA synthesis in F0 rats. Cancer Lett. I1, 221. Martin R. A., Daly A. M., DiFonzo C. J. and de la Iglesia F. A. (1984). Randomization of animals by computer program for toxicity studies. J. Am. Coll. Toxicol. 3, 1. Renwick A. G. and Sims J. (1983). Distension of the urinary bladder in rats fed saccharin containing diet. Cancer Lett. 18, 63. Rock R. C., Walker W. G. and Jennings C. D. (1986). Textbook of Clinical Chemistry. Ist Ed. p. 1254. W. B. Saunders Co., Philadelphia, PA. Schoenig G. P. and Anderson R. L. (1985). The effects of high dietary levels of sodium saccharin on mineral and water balance and related parameters in rats. Fd Chem. Toxic. 23, 465. Schoenig G. P., Goldenthal E. I., Geil R. G., Richter W. R. and Carlborg F. W. (1985). Evaluation of the dose response and in utero exposure to saccharin in the rat. Fd Chem. Toxic. 23, 475. Sims J. and Renwick A. G. (1984). Diurnal variation in the excretion of indican in rats fed saccharin containing diet. Cancer Lett. 23, 259. Tan H. S. I. and Pan P. W-C. (1982). Assay of saccharin and sodium saccharin in animal feed. J. Chromat. 238, 500. Tietz N. W., Pruden E. L. and Siggaard-Andersen O. (1986). Textbook of Clinical ChemLstry. Ist Ed. p. 1172. W. B. Saunders Co., Philadelphia, PA. United States Department of Agriculture (I 961 ). Agriculture Handbook No. 8.
0278-6915/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc
Research Section EFFECT OF S O D I U M S A C C H A R I N A N D C A L C I U M S A C C H A R I N ON U R I N A R Y P A R A M E T E R S IN RATS F E D PROLAB 3200 OR AIN-76 DIET M. J. FISHER, T. SAKATA,T. S. TIBBELS, R. A. SMITH, K. PATIL, M. KHACHAB, S. L. JOH^NSSON and S. M. COHEN* Department of Pathology & Microbiology and the Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68105, USA (Received 4 April 1988; revisions received 8 September 1988)
Abstract--The effects of the salt form of saccharin and of diet on urinary ion levels have been studied in rats. Sodium saccharin (NaS) or calcium saccharin (CaS) was fed at a level of 5% in either Agway Prolab 3200 diet or AIN-76 diet to male, 5-wk-old F344 rats for l0 wk. The AIN-76 diet contained considerably less calcium, sodium and potassium than the Prolab 3200 diet, and smaller amounts of these ions were eliminated over 24 hr in the urine of rats fed the AIN-76 diet. Although food consumption was less in the groups fed AIN-76, total urinary saccharinate ion excretion with either saccharin salt was comparable with, or even higher than, that excreted by rats fed either salt in the Prolab 3200 diet. Rats fed Prolab 3200 eliminated approximately equal amounts of saccharinate ion in the faeces and urine. Rats fed AIN-76 eliminated about 10-20 times as much saccharin in the urine as in the faeces. Total saccharin excretion (faecal and urinary) was not influenced by the salt form. Water intake and urine volume were lower in rats fed control AIN-76 diet in comparison with those fed Prolab 3200, and were increased above the control level in groups fed saccharin in the AIN-76 diet. Urine electrolyte levels and osmolality were lower in the groups fed AIN-76. In general, NaS administration in either diet resulted in increased urinary sodium compared with controls, and the pH was at, or above, the level of control rats. CaS resulted in increased urinary calcium and decreased pH. There were marked diurnal variations in the urinary excretion of the various electrolytes, pH, and urine volume over a 24-hr period in all rats. This diurnal variation was more pronounced in the rats fed the Prolab 3200 diet. These results indicate that NaS and CaS have marked effects on the excretion of urinary electrolytes, and that these effects are influenced by diet.
similar in rats fed any one of the salt forms at comparable levels in the diet. However, there were marked variations in urinary pH and sodium, potassium, calcium and other ions when the different salt forms were administered (Hasegawa and Cohen, 1986). In most of the studies in our laboratory we have used the Prolab 3200 diet (originally referred to as Charles River diet) produced by Agway Inc. (St Marys, OH, USA). In related studies on saccharin a variety of diets have been used, including Purina no. 5002 (Schoenig et al., 1985), Oriental M F (Fukushima et al., 1983) and Wayne (Lawson and Hertzog, 1981). There are differences between these diets in the levels of minerals and other nutrients and these may result in numerous differences in the levels of urinary ions excreted. The present study was designed to compare the effects in male rats of diet, as well as saccharin salt form, on urinary ion levels. Also, since the excretion of substances in the urine is dependent on levels ingested and rats generally eat at night, we considered it important to determine the variations in ion concentrations over a 24-hr period. For diet comparisons, we selected the Prolab 3200 diet and a modified AIN-76 diet. Prolab 3200 is a closed-formula diet that is cereal based, unrefined, and unpurified. AIN-76 diet was developed by the American Institute of Nutrition as a purified, openformula diet that can be formulated exactly as specified (Committee on Standards for Nutritional
INTRODUCTION Sodium saccharin (NaS) increases the proliferation rate of the urothelium of the male rat when it is administered at high doses in the diet (Cohen, 1985; Fukushima and Cohen, 1980). The salt form in which saccharin is administered plays a critical role in the response of the bladder epithelium to the compound (Anderson et al., 1988; Hasegawa and Cohen, 1986; J. Ashby, personal communication, 1988). In a short-term proliferation assay (Hasegawa and Cohen, 1986) the increase in labelling index in the bladder epithelium following potassium saccharin administration was smaller than the response to sodium saccharin. Calcium saccharin (CaS) and acid saccharin had no effect on the labelling index in the same assay. This difference in biological responsiveness to the different forms of saccharin was not due simply to differences in absorption from the gastro-intestinal tract or differences in excretion in the urine (Hasegawa and Cohen, 1986). Concentrations of the saccharinate anion in the urine were *To whom reprint requests should be addressed: Department of Pathology & Microbiology, University of Nebraska Medical Center, 42nd & Dewey Avenue, Omaha, NE 68105, USA. Abbreviations: CaS=calcium saccharin; HPLC=highperformance liquid chromatography; NaS=sodium saccharin. I FCT 2"}:I - - A
M. J. FlsrFr~ et al. Table I. Experimental design Group no.
Diet
1 2 3 4 5 6
Prolab 3200 Prolab 3200 Prolab 3200 AIN-76 AIN-76 AIN-76
Treatment Control Sodium saccharin (5%) Calcium saccharin (5%) Control Sodium saccharin (5%) Calcium saccharin (5*/,)
Studies, 1977 and 1980). There are significant differences between these two diets, including marked differences in the levels of specific minerals. The ash and mineral content of the AIN-76 diet is considerably less than that of the Prolab 3200 diet (Agway Inc., 1986; Committee on Standards for Nutritional Studies, 1977 and 1980). Semi-synthetic diets have been suggested for use in toxicology and carcinogenicity studies (discussed at the Workshop to Optimize Diet for Rodents in Chemical Carcinogenicity Studies, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 15-16 October, 1985), and therefore it is essential to study the effects of these diets on physiological parameters, such as mineral metabolism, that might influence the response to a test chemical. MATERIALS AND M E T H O D S
Weanling F344 rats (Charles River Breeding Laboratories, Inc. Kingston, NY, USA) were fed Prolab 3200 pelleted diet during a 1-wk quarantine period. At 5 wk of age, they were randomly divided into six groups of ten rats each, using the weightstratified method of Martin et al. (1984), and started on the test diets (Table 1). The rats were housed five to a cage in polycarbonate cages (16 x 18 x 20in.) with stainless-steel wire-bar covers (Lab Products, Inc., Maywood, N J, USA) and dry corn-cob bedding. They were kept at 24 + T~C and 50 + 20% relative humidity, with a 12-hr light/dark cycle. Food and tap-water were available ad lib. For each cage of rats, food and water consumption over 3-day periods were measured six times during the study. The chemical consumption for the 10wk of the experiment was estimated by extrapolation of the food-consumption measurements. Body weights were determined periodically. NaS (lot no. 053F-0655, Sigma Chemical Co., St Louis, MO, USA) or CaS (provided by Dr T. Lawson, originally synthesized by Sherwin-Williams Co., Cincinnati, OH, USA) was mixed at a level of 5% by weight into either Prolab 3200 (Agway, Inc.), or AIN-76 diet (Committee on Standards for Nutritional Studies, 1977 and 1980). In the AIN-76 diet, the addition of saccharin was compensated for by a comparable reduction in dextrin, and the diet was prepared without added antioxidant and pelleted at the Eppley Institute for Research on Cancer under the supervision of Dr Diane Birt. The saccharin salts were mixed into Prolab 3200, and the diets were pelleted by Dyets Inc. (Bethlehem, PA, USA). The diets were stored in a freezer (-20~C) until used. Prolab 3200 and AIN-76 diets containing NaS were analysed (Hazleton Laboratories America Inc., Madison, WI, USA) for pH, protein, fat, moisture, ash, crude fibre (AOAC, 1984), carbohydrates,
calories (USDA, 1961) vitamins, minerals (Dahlquist and Knoll, 1978) and specific ions (AOAC, 1984). The pH of freshly voided urine was determined at the start of the study and at the end of wk I, 2, 3, 4, 6, 8 and 10 using a microelectrode (M1-40 Microcombination pH probe, Microelectrodes Inc., Londonderry, NH, USA). For these measurements, rats were removed individually from their cage beginning at 07.00 hr, and a drop of urine was expressed from the bladder onto a glass petri dish. The microelectrode was immediately immersed in the drop and the pH was determined. During wk 4 of the experiment, three rats from each group were housed individually in stainless-steel metabolism cages that were equipped for separation of urine and faeces (Model no. Spec. LC-176, Hazleton Co., Aberdeen, MD, USA). Food and water were available ad lib. Urine was collected at 4-hr intervals over 24 hr. On days 60 and 70 urine was collected from three rats from each group kept in metabolism cages for 24 hr (10.30-10.30 hr) and 22 hr (08.304)6.30 hr), respectively. Urine was collected via a funnel connected to a 50-ml polypropylene test tube. For 4-hr collection periods, the test tubes were placed in ice-filled plastic containers to prevent fermentation of the urine, and then the urine sample s were refrigerated at 4~C until they were analysed (within 24hr). For longer collection periods, I ml toluene was added to each test tube as a preservative. Urine pH was determined using the microelectrode immediately after each collection period was completed. At the end of each collection period, the faeces were removed from the metabolism cages, weighed and frozen ( - 6 ° C ) . The volume of urine was measured and then the urine was centrifuged (2000 rpm, 850g for 5 min; Sorvall T6000, Beckman Instruments Inc., Brea, CA, USA). Urinary concentrations of sodium, potassium, calcium, creatinine and urea were determined using the Beckman Astra 4 or 8 (Beckman Instruments Inc). Sodium and potassium concentrations (Tietz et al., 1986) were evaluated by an ion-specific electrode method (valinomycin for potassium). Calcium concentration was measured by a colorimetric method using o-cresolphthalein complex ion (Fraser et al., 1986). Creatinine concentration was determined using picric acid (Rock et al., 1986). Urea concentration was determined via the change in conductivity of urease due to ammonium ion and bicarbonate ion (Rock et al., 1986). Urinary osmolality (mOsm/kg) was measured by freezing point depression (Rock et al., 1986; Advanced Digimatic Osmometer, Model 301I, Advanced Instruments, Inc., Needham Heights, MA, USA). Samples of diet, faeces and urine were analysed for saccharin using a modification of the highperformance liquid chromatographic (HPLC) procedure of Tan & Pan (1982). Samples of diet or faeces (approximately 0.5 g each) were dried overnight in a single-wall transite oven (Blue M Electric Co., Blue Island, IL, USA), prehydrated with 0.5ml 0.1M-NaOH, extracted three times with 2.5ml 0.1 M-NaOH, and centrifuged (10min, 10002000 rpm) between each extraction. The supernatants were combined in a 10-ml volumetric flask and diluted to volume with triple-distilled water. The
Urinary function in rats fed saccharin salts AIN-76 diet was heated in a boiling water-bath during each extraction with NaOH in order to precipitate solubilized proteins that would otherwise give a gelatinous solution. Urine samples (I.0ml) were placed in a 10-ml volumetric flask, and diluted to volume with 7.5 ml 0.1 M-NaOH and !.5 ml H20. A l-ml aliquot of a stock solution of 0.122 M-NaS was treated in the same manner as urine for use as an extracted standard. An aliquot (0.5 ml) of the diet or faeces extract, diluted urine sample, or standard was applied to a 3-ml disposable (Baker Bond) quaternary amine (SAX) column that had been conditioned by sequential washes with methanol, ammonium hydroxide (sp. gr, 0.9), water and 0.2 M-HCI. After application of the sample, the column was washed with two 1-ml aliquots of methanol followed by one 2-ml aliquot of water. The saccharin was eluated into a 5-ml volumetric flask with 2-ml aliquots of 0.2M-K2HPO, buffer (pH 8.8), and then diluted to volume with the same buffer. HPLC was performed on a Waters (Waters Associates Inc., Milford, MA, USA) system consisting of a model 510 pump, U6K injector (250/~l loop), temperature control module oven, and a model 481 spectrophotometer interfaced with a HewlettPackard (Avondale, PA, USA) 3390A integrator. An aliquot (100/~l) of the sample was analysed using a Waters Novapak Ci8 column (15 cm x 0.39 cm i.d.) eluted with methanol-acetic acid-water (166:4:830, by vol, pH 3.5) at a flow rate of I ml/min at 30°C with UV detection at 254 nm, 0.5 AUFs. Experimental data was evaluated statistically on an IBM 4381 computer using a Generalized Linear Model procedure from the Statistical Analysis
3
System software package (SAS Institute, Inc., Car),, NC). Differences in body weight, diet and water intake, excretion of urine and faeces, saccharin consumption and excretion, and urinary sodium, potassium and calcium, were analysed at wk 4, 8 and 10. Multiple group comparisons were made simultaneously using Duncan's method. Diurnal analysis pertains to the wk 4 data, based on measurements of six consecutive 4-hr collections. RESULTS
Analyses of diets Analyses confirmed that there are numerous differences in the composition of the Prolab 3200 and AIN-76 diets (Table 2), and that the mineral content of AIN-76 is much lower than that of Prolab 3200. The calorific values of the AIN-76 and Prolab3200 diets are 380 and 345 kcal/100 g, respectively (International Life Sciences InstituteNutrition Foundation, personal communication, 1986).
Food and water consumption Throughout the experiment, control rats fed AIN-76 (Group 4) consumed less feed than did the controls fed Prolab 3200 (Group 1) (Table 3). Despite the greater calorific value of the AIN diet (Agway Inc., 1986; Committee on Standards for Nutritional Studies, 1977 and 1980), control rats fed AIN-76 diet consumed fewer calories than did those fed Prolab 3200 (calculated values not shown). It should be noted that pelleted Prolab 3200 is a softer diet than pelleted AIN-76. Although spillage was not significant in any of the cages included in food consumption
Table 2. Analysisof Prolab3200 and AIN-76diets AIN-76 Parameter Prolab3200 meal purifieddiet pH 6.3 7.5 Protein (%) 22.8 18.3 Moisture (%) 10.6 4.9 Fat (%) 5.8 5.1 Ash (%) 6.8 2.5 Crude fibre (%) 3.7 4.1 Carbohydrates (%) 50.3 65.l Calories (kcal/100g) 345 380 Cystine(rag/g) 3.82 0.69 Methionine(mg/g) 3.73 7.83 Chloride (ppm) 5040 1790 Bromide (ppm) < 100 < 100 Phosphate (ppm) 2180 1530 Nitrate-nitrogen(ppm) < 100 < 100 Nitrate-nitrogen(ppm) < 200 < 200 Sulphate (ppm) 597 912 Ca (ppm) 12,363 4843 Mg (ppm) 2401 497 K (ppm) 9350 3880 P (ppm) 9776 4975 Na (ppm) 2841 1018 AI (ppm) 107.9 12.0 Ba (ppm) 8.41 0.15 Fe (ppm) 299 51.6 Sr (ppm) 15.8 2.08 B (ppm) 7.23 1.00 Cu (ppm) I 1.0 4.I Zn (ppm) 82.0 33.I Mn (ppm) 68.7 46.2 Cr (ppm) 0.88 0.66
4
M.J. Ftsnr~ et al.
Table 3. Diet and water intake,saccharinconsumption,and body weight of rats fed sodium saccharinor calciumsaccharin in Prolab3200 or AIN-76diet Daily intake (mean ± SEM) of: Croup no. 1 2 3 4 5 6 l 2 3 4 5 6
Diet Prolab 3200
AIN-76
Prolab 3200
AIN-76
Treatment
No. o f rats
Body weight (mean + S E M g)
Diet (g)
Water (ml)
Control NaS CaS Control NaS CaS
10 10 10 10 10 9*
225 214 207 222 199 199
+ ± ± ± + ±
Wk5 3 3° M 3 2~ 3b
19 24 30 16 16 16
_+ 0.4 ~ _+ 0.7 ~ ~ 2.4 ~ + 0.3 ± 0.9 _+ 0.2
30 33 33 22 32 42
+_ 0.6 ~ + 1.6 + 0.4 d ~_ 0.4 _+ 0.5 ~ ± 4.6 ~
Control NaS CaS Control NaS CaS
l0 10 10 l0 10 9*
276 261 260 273 247 246
Wk 8 "~-4 ± 4~ ___4 ~ _ 3 + 3~ ± 4b
18 20 23 17 15 15
_+ 0.1 ~ +_ 0.3 '~ _+ 0.1 ~ + 0.2 _+ 0.2 ± 0.5
28 34 33 22 28 28
_+ 0.4 +_ 1.5 ~ _+ 0.7 ~ + 2.7 ° ± 0.2 ~ + 0.5 b
Control NaS CaS Control NaS CaS
10 10 10 10 10 9*
298 282 279 295 266 266
+ 4 +. 4* ± 5* ± 3 ± 4b ± 4b
17 18 23 14 14 14
_+ 0.2 + 0.1 ~ ± 1.0~ + 0.2 a _+ 0.2 _+ 0.1
23 33 35 17 24 25
_ 0.3 + 1.0'~ ± 0.6 *a ± 0.2* _+ 0.5 h _+ 2.0 b
Total consumption o f saccharin/rat at wk 10 (g)
D
m
m
Wk I0 1 2 3 4 5 6
Prolab 3200
AIN-76
N a S = sodium saccharin *One rat died during the experiment. Within each parameter, values m a r k e d Significantly different from G r o u p I, Significantly different from G r o u p 4, Significantly different from G r o u p 5, Significantly different from G r o u p 6,
0 56 73 0 34 36
C a S = calcium saccharin
with superscripts differ significantly as follows: Ap = 0.05, ' P ~ 0.01, ° P = 0.001; bp = 0.001; cp = 0.001; dp = 0.001.
measurements, it is possible that slightly more of the Prolab 3200 fell from the feeders into the bedding. The possibility that consumption of Prolab 3200 has been overestimated due to spillage is borne out by the body-weight data (Table 3). Consumption of saccharin-containing AIN-76 diets (Groups 5 and 6) was similar to that of the control AIN-76 diet. In contrast, rats fed Prolab 3200 ate more CaS diet (Group 3) than NaS diet (Group 2), and both of these groups consumed more feed than the control group did. The amount of saccharin consumed during the experiment (Table 3) was greater for Groups 2 and 3 than it was for Groups 5 and 6 because of the greater consumption of the Prolab 3200 diet. More water was consumed by the control group fed Prolab 3200 compared with the control group fed AIN-76 (Table 3). With both diets, feeding either NaS or CaS increased water consumption compared with the corresponding control group. Water consumption by the saccharin-fed rats was significantly greater in the Prolab 3200 groups than in the AIN-76 groups only during wk 10. Weight gain The mean body weights of the control groups (1 and 4) did not differ significantly at wk 4, 8 or 10. Rats fed NaS or CaS in either diet gained less weight than the corresponding control group (Table 3). This decreased weight gain was more notable in rats fed the AIN-76 diet. Faecal elimination o f saccharin The amount of faeces (wet weight) voided daily gradually increased during the experiment in rats fed Prolab 3200 but not in those fed AIN-76 (Table 4).
The amount of faeces voided per day was similar in all three groups fed AIN-76. The rats fed Prolab with NaS voided approximately the same amount of faeces per day as the control group fed Prolab. However, in contrast the group fed CaS in Prolab 3200 voided significantly more faeces at all three time points in comparison with either the control or NaS groups (Table 4). Except at wk 4, the saccharin concentration was similar in the faeces of rats fed Prolab 3200 containing NaS or CaS (Table 4). In contrast, rats fed CaS in AIN-76 had higher saccharin concentrations in the faeces than did rats fed NaS in AIN-76, although the differences were significant only at wk 10. The faecal saccharin concentration was lower in both AIN-saccharin groups than in the corresponding Prolab-saccharin groups. Given these differences in concentration, along with differences in the amount of faecal material voided, the total amount of saccharin eliminated in the faeces was appreciably higher in rats fed the Prolab diet than in those fed the AIN diet (Table 4). Also, with both diets, the total amount of saccharin eliminated in the faeces was greater in the rats fed CaS than in those fed NaS; this increase was statistically significant only for the Prolab diet. Urinary excretion o f saccharin Urine volume was increased in rats fed the AIN-76 diet containing either form of saccharin (Table 4). This saccharin-associated increase in urine volume was not seen in Prolab-fed groups during the time the rats were housed in the metabolism cages. Urinary saccharin concentration was measured only at 8 and l0 wk (Table 4). For rats fed a particular basal diet, there appeared to be no differences
Urinary function in rats fed saccharin salts
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Urinary function in rats fed saccharin salts associated with saccharin salt form in either urinary saccharin concentration or in the amount of saccharin voided over 24 hr. Although the urinary concentration of saccharin in rats fed the AIN-76 diet was less than that in the rats in the corresponding groups fed Prolab 3200 (this observation was statistically significant for the CaS-fed groups at wk 8), the higher urine volume in the AIN-saccharin groups resulted in a significantly larger amount of saccharin being excreted in the urine over 24 hr by the AIN-76 groups. This contrasts with the comparatively low levels of saccharin eliminated via the faeces in the AIN-76 groups. As a result, the ratio of urinary saccharin to faecal saccharin was dramatically higher in rats fed AIN-76 diet containing saccharin compared with that in rats fed the corresponding Prolab diets (Table 4). Saccharin elimination over 24 hr by rats fed Prolab 3200 diet was almost equally divided between the urine and faeces, whereas in rats fed the AIN-76 diet, 10-20 times as much saccharin was excreted in the urine as in the faeces.
Urinary parameters--longitudinal comparisons Except for one rat which was eliminated from G r o u p 3 at the 8-wk collection, all of the total urinary creatinine levels were within normal limits. Because at 4 wk the 24-hr urine colletion was divided into six 4-hr periods, insufficient sample was available for urea analysis. No significant differences were observed between any of the groups in total urea excretion in the urine at 8 or 10 wk. However, the concentration of urea was signifiantly higher in rats fed CaS in the Prolab diet at 10 wk, but not at 8 wk. Because of the marked increase in urinary volume, the concentration of urea in the rats fed the A I N diet containing NaS or CaS was significantly less than in the control group at both time points. There were no significant differences in total urea or urea concentration between rats fed NaS or CaS in either diet. Concentrations of N a ' , K + and Ca-" in urine samples collected over 24 hr are shown in Table 5. Urinary concentrations of Na + and K ÷ were lower for the rats fed the AIN-76 diet than for the corre-
450 [--
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225 I.-
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0400
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~'\
~i
i
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0800
1200
.... 1600
2000
Time of duy
Fig. 2. Urinary sodium concentrations. Urine was collected over six consecutive 4-hr intervals. The time of day at which each interval ended is indicated. - - A - - = Group 1 (control Prolab 3200); - - O - - = Group 2 (Prolab 3200 + sodium saccharin); - - I 1 - - = Group 3 (Prolab3200+calcium saccharin); - - /~ . . . . Group 4 (control AIN-76); ---© - = Group 5 (AIN-76+sodium saccharin); [] . . . . Group 6 (AIN-76 + calcium saccharin). sponding Prolab 3200 groups. Similarly, the total levels of Na + and K + excreted in the urine over 24 hr were generally less in rats fed AIN-76 diet. The urinary concentration of Ca 2~ was also generally lower in the rats fed AIN-76 diet. Feeding either NaS or CaS increased the urinary concentration and excretion of the relevant cation in both the AIN-76 and Prolab 3200 groups. In rats fed AIN-76 containing saccharin, the urinary K + concentration was significantly less than in those fed the control AIN-76 diet; this difference was related to the difference in urine volume and, therefore, there were no differences between the groups fed AIN-76 diet in the total amount of K + excreted in the urine. The pH of the urine of groups fed the AIN-76 diets was significantly less than that of the urine of any of the groups fed Prolab 3200 (Fig. 1). Within the three AIN-76 groups, there was little difference in the urinary pH at any of the time points. The urinary pH of rats fed CaS in Prolab 3200 was significantly lower than the urinary pH of rats fed NaS in the same diet at wk 1, 2, 4, 6 and 8, and significantly lower than the Prolab controls at wk 1, 4, 6 and 10. In general, the urinary pH of the rats fed Prolab 3200 containing
8.0
./=
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I 14
I 21
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I 49
I 56
I 65
J 70
Fig. 1. pH of freshly voided urine samples collected from rats at 07.00 hr at the specified weeks of the experiment. - - A - - = Group l (control Prolab 3200); - - O - - = Group 2 (Prolab 3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . . Group 4 (control AIN-76); - - - © . . . . Group 5 (AIN-76 + sodium saccharin); - - - [ ] . . . . . Group 6 (AIN-76 + calcium saccharin).
0
0
I 0400
I 0800
I 1200
I 1600
i 2000
Time of day
Fig. 3. Urine potassium concentrations. The indicated times represent the end of a 4-hr collection period. - - A - = Group I (control Prolab 3200); - - O - - = Group 2 (Pro]ab3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - O . . . . Group 5 (AIN-76 + sodium saccharin); ---17 .... Group 6 (AIN-76 + calcium
saccharin).
M. J. FLSHr,R et al. 150
relatively constant between 20.00 and 08.00 hr, but it was decreased at 12.00 and 16.00hr. In general, the diurnal variation of the different cations in the urine was greater in the rats fed Prolab 3200 than in those fed AIN-76. However, with all of these parameters, there was significant variation • within groups at the different time points.
100
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0
DISCUSSION
I 0400
I 0800
I 1200
I '1600
I 2000
Time of roy
Fig. 4. Urine calcium concentrations. The indicted times represent the end of a 4-hr collection period. - - A - - = Group 1 (control Prolab3200); - - O - - = G r o u p 2 (Prolab 3200 + sodium saccharin); - - m - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - O = Group 5 (AIN-76 + sodium saccharin); - - - [ 3 - - = Group 6 (AIN-76+ calcium saccharin). NaS was similar to the urinary pH of rats fed the control Prolab diet. Urine osmolality was similar among the three groups fed Prolab 3200, whereas the urine from rats fed the AIN-76 diet containing NaS or CaS had a lower osmolality than the urine from the controls. The osmolality of the urine from control AIN-76 rats was less than that of urine from rats fed the control Prolab diet. D i u r n a l variations
The diurnal variations within urinary pH and concentrations of Na ÷, K ÷ and Ca 2+ are shown in Figs 2-5. Urinary concentrations of Na ÷ and K + were generally higher at night than during the day, with the lowest values usually occurring at 16.00 or 20.00hr. Urinary Ca 2÷ remained at approximately the same level throughout the 24-hr period except for an increase in concentration at 16.00 hr. Urinary pH was measured on freshly voided samples, rather than on collected samples (Fig. 5). There was less diurnal variation for the rats fed the different AIN-76 diets than for those fed the corresponding Prolab 3200 diets. In the rats fed Prolab 3200, the urinary pH was 8.0 7.5
Z el
Z /Z.
7.0 6.5 6.0 5.5
5.C
I 0400
I oeoo Time
I 1200 of
I 1600
I 2000
doy
Fig. 5. pH freshly voided urine from rats not contained in metabolism cages. - - A - - = Group I (control Prolab 3200); - - 0 - - = Group 2 (Prolab 3200 + sodium saccharin); - - I I - - = Group 3 (Prolab 3200 + calcium saccharin); - - - A . . . . Group 4 (control AIN-76); - - - © . . . . . . Group 5 (AIN-76 + sodium saccharin); ---I-l . . . . Group 6 (AIN-76 + calcium saccharin).
The present experiment has confirmed the previous finding that rats fed NaS or CaS excrete similar levels of saccharin in the urine (Hasegawa and Cohen, 1986; Patricia Kraft, personal communication 1984). However, despite this similarity, there appear to be marked differences in urinary pH and the excretion of various electrolytes in the urine following administration of these two saccharin salt forms. These differences have been more clearly defined in the present study, as has the influence of administering NaS or CaS in two very different diets. Since the AIN-76 diet has more calories per gram than does the Prolab 3200 diet (International Life Sciences Institute-Nutrition Foundation, personal communication 1986), rats would be expected to consume less of the AIN-76 diet than the Prolab diet. Also, with saccharin present in the diet at such high levels, the rats would be expected to eat slightly greater amounts of the saccharin-containing diets than the control diets in order to compensate for the lower number of calories per gram of diet. Essentially this was observed in the present experiment, although the consumption of Prolab 3200 was even greater than would have been expected. It is possible that spillage of the feed contributed to the higher values of food consumption of Prolab 3200, since the groups fed control AIN-76 and Prolab 3200 gained weight at similar rates. For both CaS and NaS total, the dose of saccharin, calculated from data on food consumption and diet analyses, was less for rats fed the AIN-76 diet than for those fed the Prolab 3200 diet. A marked increase in urine volume was seen following administration of NaS or CaS in the AIN-76 diet, but there was no such increase with the Prolab 3200 diet (Table 4). The urinary saccharin concentration was similar regardless of the salt form or diet in which the saccharin was administered. The total amount of saccharin voided over 24 hr was not affected by saccharin salt form, but values for the rats fed AIN-76 were higher than those for rats fed Prolab3200 due to the increase in urine volume. Marked differences in the faecal excretion of saccharin were observed, and these were consistent with the differences in saccharin intake (Table 4). Rats fed AIN-76 diet excreted smaller amounts of faeces with lower concentrations of saccharin than did rats fed the corresponding Prolab 3200 diets. These findings with the Prolab 3200 diet are similar to those previously reported in rats fed NaS in Purina diet (Schoenig and Anderson, 1985). The AIN-76 diet has a lower mineral content than the Prolab 3200 diet (Table 2), and the rats fed AIN-76 excreted urine containing smaller amounts of Na ~, K + and Ca :+ than did those fed Prolab 3200. Of particular note was the consistently lower urinary pH observed in rats fed the control and saccharin-
Urinary function in rats fed saccharin salts containing AIN-76 diets, in contrast to the approximately neutral to slightly alkaline pH of the urine of rats fed Prolab 3200. The urinary pH of Prolab 3200fed rats increased slightly following administration of NaS and decreased following CaS administration. As would be anticipated, urinary Na ~ was considerably elevated in rats fed NaS, whether in the Prolab 3200 diet or AIN-76 diet; similarly, urinary Ca `'+ was elevated in rats fed CaS in either of the diets. Numerous studies (deGroot et al., 1988; Fukushima et al., 1986; Harguindey, 1982; Kritchevsky, 1986; Renwick and Sims, 1983; Schoenig and Anderson, 1985) have indicated an influence of pH, levels of sodium and calcium, urine volume, and calorie intake on the biological response to a variety of chemicals. Thus, it is important to study the levels of various ions in the urine or target tissues of rats fed high levels of various compounds in the diet. It is also essential to note that the time of observation of the various parameters is particulary important because of the striking diurnal variation in the urinary ion excretion (Sims and Renwick, 1984). There is marked variation in the urinary excretion of the various electrolytes, and in pH and urinary volume, over a 24-hr period, as would be anticipated considering the eating and drinking habits of the rat. We also observed that measurement of urinary pH is more accurate if done at the same time(s) each day, and carried out immediately using a microelectrode, rather than on collected specimens. Collected specimens vary considerably in pH over time. The present studies indicate that, although the level of saccharinate ion in the urine is similar, there are marked differences in the excretion of various ions (Figs 1-3; Table 2) by rats fed NaS in comparison with those fed CaS. Also, administering these compounds in two very different diets, Prolab 3200 and the AIN-76, results in marked differences in the excretion of various ions in the urine, although the saccharinate excretion appears similar. Acknowledgements--We gratefully acknowledge the advice and comments of Drs Emily Garland, Diane Birt, Gerald Schoenig and Jeffrey Huth, the technical assistance of Margaret St. John and James Gulizia, and the assistance of Jan Leemkuil in the preparation of this manuscript. This work was supported in part by a grant from the International Life Sciences Institute Nutrition Foundation, and by USPHS grant no. CA36727 from the National Cancer Institute. REFERENCES
Agway Inc. (1986). Prolab formulas for precise feeding of rats, mice, and hamsters. Agway Inc., St Mary's, OH, USA. Anderson R. L., Lefever F. R. and Maurer J. K. (1988). The effect of various saccharin forms on gastrointestinal tract, urine and bladder of male rats. Fd Chem. Toxic. 26, 665. AOA,S (1984). Official Methods of Analysis. Edited by S. Williams. 14th Ed. Association of Official Analytical Chemists, Arlington, VA, USA.
Cohen S. M. (1985). Multi-stage carcinogenesis in the urinary bladder. Fd Chem. Toxic. 23, 521. Committee on Standards for Nutritional Studies (1977). Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 107, 1340. Committee on Standards for Nutritional Studies (1980). Second report of the Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 110, 1726. Dahlquist R. L. and Knoll J. W. (1978). Inductively coupled plasma-atomic emission spectrometry: analysis of biological materials and soils for major, trace, and ultra-trace elements. Appl. Spectrosc. 32, 1. deGroot A. P., Feron V. J. and Imnael H. R. (1988). Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/carcinogenicity testing. Fd Chem. Toxic. 26, 425. Fraser D., Jones G., Kooh S. W. and Radde I. C. (1986). Textbook of Clinical Chemistry. Ist Ed. p. 1317. W. B. Saunders Co., Philadelphia, PA. Fukushima S., Arai M., Nakanowatari J., Hibino T., Okuda M. and lto N. (1983). Differences in susceptibility to sodium saccharin among various strains of rats and other animal species. Gann 74, 8. Fukushima S. and Cohen S. M. (1980). Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res. 40, 734. Fukushima S., Shibata M-A., Shirai T., Tamano S. and Ito N. (1986). Roles of urinary sodium ion concentration and pH in promotion by ascorbic acid of urinary bladder carcinogenesis in rats. Cancer Res. 46, 1623. Harguindey S. (1982). Hydrogen ion dynamics and cancer: an appraisal. Med. Pediat. Oncol. 10, 217. Hasegawa R. and Cohen S. M. (1986). The effect of different salts of saccharin on the rat urinary bladder. Cancer Lett. 30, 261. Kritchevsky D. (1986). Diet, nutrition, and cancer: the role of fiber. Cancer, N.Y. 58, 1830. Lawson T. A. and Hertzog P. J. (1981). The failure of chronically administered saccharin to stimulate bladder epithelial DNA synthesis in F0 rats. Cancer Lett. I1, 221. Martin R. A., Daly A. M., DiFonzo C. J. and de la Iglesia F. A. (1984). Randomization of animals by computer program for toxicity studies. J. Am. Coll. Toxicol. 3, 1. Renwick A. G. and Sims J. (1983). Distension of the urinary bladder in rats fed saccharin containing diet. Cancer Lett. 18, 63. Rock R. C., Walker W. G. and Jennings C. D. (1986). Textbook of Clinical Chemistry. Ist Ed. p. 1254. W. B. Saunders Co., Philadelphia, PA. Schoenig G. P. and Anderson R. L. (1985). The effects of high dietary levels of sodium saccharin on mineral and water balance and related parameters in rats. Fd Chem. Toxic. 23, 465. Schoenig G. P., Goldenthal E. I., Geil R. G., Richter W. R. and Carlborg F. W. (1985). Evaluation of the dose response and in utero exposure to saccharin in the rat. Fd Chem. Toxic. 23, 475. Sims J. and Renwick A. G. (1984). Diurnal variation in the excretion of indican in rats fed saccharin containing diet. Cancer Lett. 23, 259. Tan H. S. I. and Pan P. W-C. (1982). Assay of saccharin and sodium saccharin in animal feed. J. Chromat. 238, 500. Tietz N. W., Pruden E. L. and Siggaard-Andersen O. (1986). Textbook of Clinical ChemLstry. Ist Ed. p. 1172. W. B. Saunders Co., Philadelphia, PA. United States Department of Agriculture (I 961 ). Agriculture Handbook No. 8.