Dietary Ammonium Chloride for the Acidification of Mouse Urine

Abstract

A novel therapeutic compound was found to induce bladder tumors in male rats. Given the location of the tumors and the increased amounts of calcium- and magnesium-containing solids found in the urine of treated animals, we hypothesized that tumorigenesis was secondary to urine crystal formation rather than a direct effect of the drug on urothelium. To investigate the basis for the response, a method of acidifying rodent urine was needed. This study tested the efficacy of 1% dietary NH₄Cl in reducing the urinary pH of male mice. After 1 wk, urinary pH (mean ± SD) at 1 h after light onset was 7.51 ± 0.32 among controls compared with 6.21 ± 0.31 for the NH₄Cl-fed group. After 2 wk of supplementation, urinary pH was 7.78 ± 0.41 for controls and 6.20 ± 0.30 for the NH₄Cl-fed group. To investigate whether the time of collection altered urinary pH, samples also were collected 8 h after the start of the light cycle on the day of the 2-wk collection. Urinary pH was 7.12 ± 0.28 for the control group and 5.80 ± 0.23 for the NH₄Cl-fed mice. The pH differences between control and NH₄Cl-fed groups and the differences in pH within groups at 1 and 8 h were statistically significant. Dietary NH₄Cl is an effective urinary acidifier for mice. When evaluating the pH of mouse urine, care should be taken to compare samples collected at the same time after the start of the light cycle.

Numerous rodent carcinogenicity studies are performed every year around the world, as required by the US Food and Drug Administration, the European Medicines Agency, and other governmental authorities. These studies are critical to the development and marketing of new medicines for both humans and animals. During routine carcinogenicity studies in rats and mice, a novel therapeutic compound was found to induce transitional cell carcinomas in the urinary bladders of male rats.³ Because the tumors were located predominantly within the ventral half of the bladders and because treated animals had increased amounts of calcium- and magnesium-containing solids in their urine, we hypothesized that tumorigenesis was caused by epithelial irritation secondary to urine crystal formation rather than a primary effect of the drug itself. Other compounds including melamine,⁹ uracil,⁷ silicates,^4,10 sodium saccharin,¹ sodium ascorbate,¹ sulfonamides,⁸ and carbonic anhydrase inhibitors⁸ have been shown to induce urothelial tumors by this mechanism. Urinary pH can have a profound effect on the formation of urinary solids, and the formation of endogenous calcium and magnesium salts in rat urine is facilitated at a urinary pH greater than 6.5.¹ To investigate our hypothesis and the basis for the species-specificity of the observed tumorigenesis in rats, a simple method of acidifying both rat and mouse urine was needed to decrease urinary crystal formation in long-term rodent studies.

Ammonium chloride has been used to acidify urine in a variety of species including cats, humans, cattle, horses, sheep, dogs, and goats.¹¹ Although NH₄Cl has been added to water bottles to acidify the urine of mice,^5,6 automatic watering systems are typically used for long-term studies with large numbers of mice. A more convenient method of administering NH₄Cl was needed to investigate tumor formation in our setting. The present study tested the efficacy of adding 1% NH₄Cl to a standard rodent diet for urinary acidification.

Materials and Methods

This study was conducted at Bristol-Myers Squibb's AAALAC-accredited animal facility (Mount Vernon, IN) and was approved by the Mount Vernon Animal Care and Use Committee. Twenty male, 9 to 10 wk old, specific pathogen-free, Crl:CD-1(ICR)BR mice were purchased from Charles River Laboratories (Kingston, NY). The source colony was tested routinely and was negative for Sendai virus, pneumonia virus of mice, mouse hepatitis virus, mouse parvovirus, minute virus of mice, Theiler mouse encephalomyelitis virus, reovirus 3, epizootic diarrhea of infant mice virus, Mycoplasma pulmonis, Ectromelia, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, murine cytomegalovirus, murine T-cell lymphotrophic virus, Helicobacter spp., cilia-associated respiratory bacillus, Corynebacterium kutscheri, Klebsiella pneumoniae, fur mites, and pinworms. Because of the short duration of the study, the mice were not screened for subclinical pathogens after arrival, but none of the pathogens listed had been detected through the routine screening of sentinels from other groups of rodents in our facility. Our routine sentinel testing program includes quarterly testing of stock rodents and, for all studies of 1 mo or more in duration, prestudy testing, quarterly testing while on study, and end-of-study testing.

The mice were divided randomly into 2 groups and acclimated to individual housing in wire-bottomed cages (Lab Products, Seaford, DE). Noncontact pan liners under the cages (Harlan-Teklad, Madison, WI) were replaced 3 times weekly, and the cages were changed every other week. Synthetic bone chew toys (Nylabone Products, Neptune, NJ) were provided for enrichment, a 12:12-h light:dark cycle was used, and water purified by reverse osmosis was provided ad libitum through an automated watering system (Edstrom Industries, Waterford, WI).

One group of 10 mice was fed a standard rodent diet, (8728C, Harlan Teklad), and 10 mice were given the same diet manufactured with 1% NH₄Cl added (3028C, Harlan Teklad). After 1 wk of receiving either the NH₄Cl-added diet or the normal rodent diet, freshly voided urine samples were collected from each animal 1 h after the start of the light cycle.

Immediately on removal of a mouse from its cage, a culture dish was placed under the mouse. In most cases, the mice urinated as soon as they were restrained. If the mouse did not urinate immediately, gentle digital pressure was applied to the caudal abdomen to induce urination. The culture dish then was tilted and gently tapped to pool the urine along the edge of the dish. Urine pH was measured directly (sympHony SB20 m, VWR International, West Chester, PA). Only a drop or 2 (approximately 50 µL) of freshly voided urine was needed for measurement.

Because urine pH cycles naturally throughout the day, urine was collected from each animal 1 and 8 h after the start of the light cycle after 2 weeks of NH₄Cl supplementation. Urine pH was measured as described earlier. Group mean urine pH measurements were compared with the Student test by using a commercially available software program (JMP 6.0.3, SAS Institute, Cary, NC).

Results

Tables 1 and 2 show individual values for the pH of freshly voided urine from control mice and mice fed the acidifying diet for 1 wk (samples collected at 1 h after the start of the light cycle) and 2 wk (samples collected at 1 and 8 h after the start of the light cycle), respectively. The mean urinary pH of mice fed the acidifying diet was significantly (P < 0.01) lower than that of concurrent control mice at both intervals and collection time points. Urine collected from both control and NH₄Cl-fed mice 8 h after the start of the light cycle was more acidic (P < 0.01) than urine collected 1 h after the start of the light cycle.

Table 1.

pH of Mouse urine after 1 wk of dietary supplementation with 1% NH₄Cl

Control mice	NH4Cl-fed mice
7.44	6.00
7.26	5.99
7.11	5.84
7.55	Quantity insufficient
7.74	6.01
6.97	6.05
7.72	6.21
7.66	6.42
7.69	6.74
7.98	6.60
Mean ± SD, 7.51 ± 0.32	Mean ± SD, 6.21 ± 0.31a

Control mice received a standard rodent diet; experimental mice received a standard rodent diet containing 1% NH₄Cl. Animals were maintained on the respective diets for 1 wk before pH levels were measured 1 h after onset of the light phase of the photoperiod.

^aSignificant (P < 0.01) difference between levels for NH₄Cl-fed and control mice

Table 2.

pH of mouse urine after 2 wk of dietary supplementation with 1% NH₄Cl

1 h after light onset		8 h after light onset
Control mice	NH4Cl-fed mice	Control mice	NH4Cl-fed mice
8.05	Quantity insufficient	7.61	5.87
8.53	5.90	Quantity insufficient	5.73
7.93	5.89	7.01	6.01
7.08	6.26	6.85	Quantity insufficient
7.33	Quantity insufficient	Quantity insufficient	5.88
Quantity insufficient	6.73	Quantity insufficient	5.41
7.83	6.13	7.24	5.86
7.85	6.21	7.22	5.65
7.74	6.52	7.12	6.18
7.72	5.96	6.78	5.64
Mean ± SD, 7.78 ± 0.41	Mean ± SD, 6.20 ± 0.30a	Mean ± SD, 7.12 ± 0.28b	Mean ± SD, 5.80 ± 0.23ab

Control mice received a standard rodent diet; experimental mice received a standard rodent diet containing 1% NH₄Cl. Animals were maintained on the respective diets for 2 wk before pH levels were measured 1 h and 8 h after onset of the light phase of the photoperiod

^aSignificant (P < 0.01) difference between values for NH₄Cl-fed and control mice at the same time point

^bSignificant (P < 0.01) difference between samples collected at 1 and 8 h

Discussion

At 1 and 2 wk, the mean urinary pH of mice fed the 1% NH₄Cl-added diet was significantly lower than that of control mice. This finding confirmed the efficacy of 1% dietary NH₄Cl in acidifying mouse urine. By having 1% NH₄Cl mixed into the feed by the manufacturer instead of using the previously published method of adding 1% NH₄Cl to water bottles, additional labor was not needed to conduct the acidification studies. No special equipment or handling was required, and the cost of having 1% NH₄Cl added to the diet was not excessive.

At week 2, for both NH₄Cl-fed and control mice, samples measured 8 h after the start of the light cycle were significantly more acidic than samples measured 1 h after the start of the light cycle. This finding reflects the normal diurnal variation of urine pH in mice due to their primarily nocturnal consumption of food.¹ Investigators concerned with the pH of freshly voided mouse urine should take care to compare samples measured at the same point after the start of the light cycle.

The formation of crystals and sedimentation of solids in the urine increase urinary bladder tumors in rodents,^1-10 which are more susceptible to both the formation and the carcinogenic effects of calculi than are humans.² Acidification of the urine decreases the formation of urine solids and, therefore, decreases tumor formation.^3,5,6 Factors other than pH also affect the formation of solids or calculi in the urine and include changes in water consumption, the administration of renally excreted test articles, and dietary mineral intake.²

The use of 1% NH₄Cl-containing feed provides a simple, cost-effective mechanism for acidifying the urine of large groups of mice in long-term studies. Urinary calculi occasionally form spontaneously in the kidneys and bladder of individual rodents. However, to treat an individual animal, adding ammonium chloride to the animal's water bottle likely would be simpler than using a special diet.