44,567-57Of1978)
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Urinary
Calcium
Excretion
by Neomycin-Treated
LESTERM.CRAWFORD'ANDRICHARD Division
of Veterinary
Research,
Received
Food and Drug
H. TESKE
Administration,
July 28, 1977; accepted
Dogs
Beltsville,
Maryland
20705
Nouember4.1977
Urinary Calcium Excretion By Neomycin-Treated Dogs. CRAWFOKD. L. M., AND TESKE, R. H. (1978). Toxicol. Appl. Pharmacol. 44. 567-570. Two groups of adult beagle dogs were treated with neomycin sulfate iv at doses of 11 and 22 mgikg. Urine volume and calcium excretion were monitored. Serum calcium was determined in the dogs given the higher dose of antibiotic. Urinary calcium excretion was increased in both dosage groups. while urine volume increased only in the dogs given 22 mg/kg of neomycin. Neomycin had no effect on serum calcium‘in either of the two groups of treated dogs. It is postulated that the antibiotic complexed tubular calcium, thus preventing active reabsorption of the mineral in the proximal tubule. where two thirds of the filtered calcium load is normally reabsorbed. The trend toward diuresis may be due to tubular retention of equimolar amounts of water by the complexed calcium. On the basis of these results. caution is advised in treating with neomycin calcium~compromised animals such as postpartum cows and bitches.
Aminoglycoside antibiotics have been shown to lower serum calcium concentrations in cows (Crawford et al., !977). Dihydrostreptomycin administered iv to nonlactating cows resulted in a 19% reduction in total serum calcium. A similar effect was observed with neomycin in the postpartum cow and with gentamicin in an in vitro system. In all of these studies (Crawford et al., 1977) reduction of total serum calcium occurred at the expense of the bound calcium fraction. The scope of the antibiotic-induced hypocalcemia study did not permit an accounting for the lost calcium. A hypothesis was advanced that the antibiotics complexed with the calcium, thus facilitating urinary excretion. The purpose of the present study was to test that hypothesis by measuring urinary calcium concentrations, urine volume, and serum calcium concentrations in neomycin-treated dogs.
METHODS In one experiment, 20 l-year-old male dogs were selected from the beagle colony at the Division of Veterinary Research. The dogs had all been whelped in the colony, were healthy, and had no previous history of antibiotic treatment. They were fed a standard dry dog food diet and were given water ad libitum throughout the test period. Five days prior to the test period, the 20 dogs were placed into separate metabolism cages in a ’ During Veterinary
the time this work was performed (1975-1976) Dr. Crawfcrd Medicine, University of Georgia, Athens, Georgia 30602. 567
was on leave from the College
of
O,M I ~OOXX,7X:0343~~0ih7$0?.0r)~(I Copyrlghr ‘C 197X by Academic Prc\\. Inc. All rights of repruductwn in any form reserved Printed m Great Bntam
568
CRAWFORD
AND
TESKE
mechanically ventilated building. Urine receptacles were covered with fine mesh screen wire and lined with plastic to prevent contamination. Urine was collected twice daily. For the 4-day determinations, the 20 dogs were randomly divided into 2 groups of 10 each. At 1 PM on Days l-4, each dog was catheterized and 30 ml of urine was collected and frozen. The calcium concentration of these samples was determined by atomic absorption spectrophotometry using a simple dilution of urine with lanthanum to correct for phosphate interference in an air-acetylene flame. These values obtained were multiplied by the daily urine output (voided volume + 30 ml) and divided by body weight to yield milligrams of calcium per kilogram per day. At 9 AM on Day 2 and again on Day 3, one group of 10 animals was given 11 mg/kg of neomycin sulfate* iv. A second experiment was conducted using the same design as the first with the following exceptions: (1) Half of the dogs in each group were female; (2) Urinary calcium was determined on a sample taken from the individual total daily urine volume. The animals were not catheterized; (3) The dosage of neomycin sulfate was doubled-22 mg/kg rather than 11 mg/kg; (4) Control animals were injected with sterile isotonic saline; and (5) Serum calcium was determined (Loken et al., 1960) on 10 ml of blood taken daily from both the control and treated dogs. Blood was drawn from the treated animals 4 hr after administration of neomycin. Blood also was obtained from control dogs at that time of day (approximately I:00 PM). RESULTS
Student’s t test was used to compare the differences in mean daily urine calcium volume between treatment and control animals on a day-by-day basis (Tables 1 and 2) and also to compare urine production (Table 3). TABLE EFFECT
Dose (mg/kg)b 0 11
0 22
OF NEOMYCIN
ON URINARY
Day 1 0.56 0.95 0.73 0.69
2 & + +
0.52 0.65 0.66 0.44
1 CALCIUM
Day 2’ 0.84 1.91 0.63 0.76
a The data are expressed as milligrams per kilogram, b There were 10 dogs in each group. (- Neomycin was administered on Days 2 and 3. d Results are significant at a level ofp 5 0.025.
+ i & +
0.46 0.47d 0.49 0.53
EXCRETION
IN
Day 3c 0.89 1.96 0.37 1.48
? k & *
0.64 1.33d 0.17 0.59d
DOGP
Day 4 0.63 1.06 0.51 0.71
i 2 + *
0.28 0.58d 0.35 0.30
mean + SD.
In the first experiment, when 11 mg neomycin/kg was administered to the dogs, urinary calcium excretion was significantly increased on both treatment days (Days 2 and 3) and on one succeedingday (Day 4) (Table 1). Calcium excretion at the higher antibiotic dose did not follow the expected doseresponse relationship. Statistically significant urinary calcium excretion occurred only on Day 3 in this group. The consistent urinary excretion pattern in the group given 11 * Mycifradin
sulfate, The Upjohn
Co., Kalamazoo,
Michigan.
CA
EXCRETION
BY NEOMYCIN-TREATED
569
DOGS
mg/kg tended to verify the assumption that neomycin promotes urinary calcium excretion. Urine volume was not significantly affected at the 11-mg/kg antibiotic dose (Table 2). With 22 mg/kg as a dose, control urine volume progressively decreased.This made the resultsdifficult to interpret. TABLE 2 EFFECT
Dose(mg/kg)b
OF NEOMYCIN
ON URINE
Day 1
Day 2’
+ 8.1 24.1 f- 8.0 23.9 t 8.1
0 11
24.2 24.9 20.8 24.2
24.7
0 22
24.3
i
VOLUME
10.3
’ The data are expressed as milliliters per kilogram, b There were 10 dogs in each group. ’ Neomycin was administered on Days 2 and 3. d Results are significant at a level ofp I 0.005.
OF DOGP
Day 3’
t 8.0 k 9.8 f 9.3
Day 4 22.6 + 11.0 23.9 + 9.5 16.7 * 8.0 21.4 5 21.3d
24.5 & 9.1 24.8 + 1.6 15.8 + 4.2 29.8 k 23.1d
& 11.0 mean t SD.
TABLE 3 EFFECTOFNEOMYCINONSERUMCALCKJMOF DOGP Total Ca Dayb 1 2 3 4 5
Control 10.30 10.21 8.14 8.33 10.16
t + + k k
0.38 0.40 0.31 0.32 0.38
UnboundCa
Treated 9.77 9.62 8.14 8.61 9.84
31 0.49 k 0.41 f 0.33 k 0.35 f 0.44
Control 6.04 6.03 4.77 5.21 5.91
+ 0.60 3~ 0.60 i 0.41 k 0.45 k 0.60
BoundCa
Treated 6.29 5.76 4.91 5.50 5.98
+ + k 5 &
0.63 0.44 0.39 0.45 0.45
Control 4.15 k 4.18 rf3.38 k 3.12 & 4.25 &
0.81 0.88 0.66 0.51 0.89
Treated 3.56 3.76 3.23 3.11 3.82
k k t f +
0.60 0.59 0.55 0.68 0.65
” The data are expressed as milligrams per 100 milliliters, mean + SD. on Days 2 and 3. b Neomycin. 22 mg/kg. was administered
There were no differences in serum calcium concentrations between the control animals and those given 22 mg/kg of neomycin (Table 3). This finding contrasts with the results of bovine studies (Crawford et al., 1977) in which antibiotic administration produced hypocalcemia. This difference may be because4 hr elapsedbetween injection of the antibiotic and drawing of blood for calcium analysis in the present study, whereas in the bovine study blood sampleswere taken immediately after administration of the antibiotic. Perhaps mobilization of calcium into the blood stream from body stores had already compensated for the enhanced calcium excretion by the time the blood was drawn from the dogs. DISCUSSION The aminoglycoside seriesof antibiotics, including neomycin, has beenshown to bind calcium in a manner similar to that of ethylenediaminetetraacetic acid (EDTA) (Adams, 1975; Crawford and Bowen, 1971; Weinstein, 1965). It is also known that
570
CRAWFORD
AND
TESKE
EDTA promotes urinary calcium excretion by interfering with the active reabsorption of calcium in the proximal renal tubule (Simesen, 1970). Calcium complexed with EDTA is not ionized and thus is not susceptibleto the active reabsorption processes which are sensitive only to free ionized calcium. Passive reabsorption of calcium does occur in the loop of Henle and the collecting duct, but two thirds of the filtered load is reabsorbed in the proximal tubule (Koshanpour, 1976). Thus, it appears most probable that EDTA exerts its calcium excretory effect in that portion of the nephron. Neomycin enhancementof calcium excretion probably proceedsin a similar manner. The trend toward diuresis observed in the present experiment is perhaps related to increased urinary calcium. The tubular reabsorption of calcium is proportional to that of water (Simesen, 1970). Thus, prevention of tubular reabsorption of calcium results in the tubular retention of equimoiar amounts of water. This action is analogous to the action of those proprietary diuretic agentsthat act by obstructing sodium reabsorption in the proximal tubule (Levine, 1973). The findings of the present study tend to support the assumption that neomycin enhances the renal excretion of calcium (Koshanpour, 1976). On the basis of this finding, and becauseof the more immediate danger of neuromuscular blockade (Adams, 1975), calcium-compromised animals such as postpartum cows and bitches probably should not be treated with parenteral neomycin. ACKNOWLEDGMENTS
The authorsthank Patty Long andBruce Olcott for technicalsupport. The authors also acknowledgethe advice of Jerry Mitchell, Laboratory of Chemical Pharmacology,National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland. REFERENCES ADAMS, H. R. (1975). Acute adverse 987. CRAWFORD, L. M., AND BOWEN, J.
Am. J. Vet.Res.32, 357-359. CRAWFORD, L. M., CAMPBELL,
effects
M. (1971).
of antibiotics. Calcium
J.
Am. Vet. Med. Assoc. 166, 983-
binding
as a property
of kanamycin.
D. L., AND HARVEY, T. (1977). Hypocalcemic effect of aminoglycoside antibiotics in the dairy cow. Canad.J. Camp.Med. 41, 25 l-256. KOSHANPOUR, E. (1976). Renal Physiology: Principles and Functions. pp. 91, 244. W. B. Saunders, Philadelphia, Pa. LEVINE, R. R. (1973). Pharmacology: Drug Actions and Reactions.p. 285. Little, Brown, Boston, Mass. LOKEN, H. F., HAVEL, R. J., GORDON, G. S., AND WHITTINGTON, S. L. (1960). Ultracentrifugal analysis of protein-bound and free calcium in human serum. J. Biol. Chem.235, 3654-3658. SIMESEN, M. G. (1970). Calcium,inorganicphosphorus, and magnesium in healthand disease. In.Clinical Biochemistryof DomesticAnimals. (J. J. Kaneko and C. E. Cornelius, eds.), Vol. 1,2nd ed.,p. 318.AcademicPress,New York. WEINSTEIN, L. (1965). Chemotherapy of microbial diseases. In The PharmacologicalBasisof Therapeutics.(L. S. Goodman and (3. Gilman, eds.), 3rd ed., p. 123 1. Macmillan, New York.
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Urinary
Calcium
Excretion
by Neomycin-Treated
LESTERM.CRAWFORD'ANDRICHARD Division
of Veterinary
Research,
Received
Food and Drug
H. TESKE
Administration,
July 28, 1977; accepted
Dogs
Beltsville,
Maryland
20705
Nouember4.1977
Urinary Calcium Excretion By Neomycin-Treated Dogs. CRAWFOKD. L. M., AND TESKE, R. H. (1978). Toxicol. Appl. Pharmacol. 44. 567-570. Two groups of adult beagle dogs were treated with neomycin sulfate iv at doses of 11 and 22 mgikg. Urine volume and calcium excretion were monitored. Serum calcium was determined in the dogs given the higher dose of antibiotic. Urinary calcium excretion was increased in both dosage groups. while urine volume increased only in the dogs given 22 mg/kg of neomycin. Neomycin had no effect on serum calcium‘in either of the two groups of treated dogs. It is postulated that the antibiotic complexed tubular calcium, thus preventing active reabsorption of the mineral in the proximal tubule. where two thirds of the filtered calcium load is normally reabsorbed. The trend toward diuresis may be due to tubular retention of equimolar amounts of water by the complexed calcium. On the basis of these results. caution is advised in treating with neomycin calcium~compromised animals such as postpartum cows and bitches.
Aminoglycoside antibiotics have been shown to lower serum calcium concentrations in cows (Crawford et al., !977). Dihydrostreptomycin administered iv to nonlactating cows resulted in a 19% reduction in total serum calcium. A similar effect was observed with neomycin in the postpartum cow and with gentamicin in an in vitro system. In all of these studies (Crawford et al., 1977) reduction of total serum calcium occurred at the expense of the bound calcium fraction. The scope of the antibiotic-induced hypocalcemia study did not permit an accounting for the lost calcium. A hypothesis was advanced that the antibiotics complexed with the calcium, thus facilitating urinary excretion. The purpose of the present study was to test that hypothesis by measuring urinary calcium concentrations, urine volume, and serum calcium concentrations in neomycin-treated dogs.
METHODS In one experiment, 20 l-year-old male dogs were selected from the beagle colony at the Division of Veterinary Research. The dogs had all been whelped in the colony, were healthy, and had no previous history of antibiotic treatment. They were fed a standard dry dog food diet and were given water ad libitum throughout the test period. Five days prior to the test period, the 20 dogs were placed into separate metabolism cages in a ’ During Veterinary
the time this work was performed (1975-1976) Dr. Crawfcrd Medicine, University of Georgia, Athens, Georgia 30602. 567
was on leave from the College
of
O,M I ~OOXX,7X:0343~~0ih7$0?.0r)~(I Copyrlghr ‘C 197X by Academic Prc\\. Inc. All rights of repruductwn in any form reserved Printed m Great Bntam
568
CRAWFORD
AND
TESKE
mechanically ventilated building. Urine receptacles were covered with fine mesh screen wire and lined with plastic to prevent contamination. Urine was collected twice daily. For the 4-day determinations, the 20 dogs were randomly divided into 2 groups of 10 each. At 1 PM on Days l-4, each dog was catheterized and 30 ml of urine was collected and frozen. The calcium concentration of these samples was determined by atomic absorption spectrophotometry using a simple dilution of urine with lanthanum to correct for phosphate interference in an air-acetylene flame. These values obtained were multiplied by the daily urine output (voided volume + 30 ml) and divided by body weight to yield milligrams of calcium per kilogram per day. At 9 AM on Day 2 and again on Day 3, one group of 10 animals was given 11 mg/kg of neomycin sulfate* iv. A second experiment was conducted using the same design as the first with the following exceptions: (1) Half of the dogs in each group were female; (2) Urinary calcium was determined on a sample taken from the individual total daily urine volume. The animals were not catheterized; (3) The dosage of neomycin sulfate was doubled-22 mg/kg rather than 11 mg/kg; (4) Control animals were injected with sterile isotonic saline; and (5) Serum calcium was determined (Loken et al., 1960) on 10 ml of blood taken daily from both the control and treated dogs. Blood was drawn from the treated animals 4 hr after administration of neomycin. Blood also was obtained from control dogs at that time of day (approximately I:00 PM). RESULTS
Student’s t test was used to compare the differences in mean daily urine calcium volume between treatment and control animals on a day-by-day basis (Tables 1 and 2) and also to compare urine production (Table 3). TABLE EFFECT
Dose (mg/kg)b 0 11
0 22
OF NEOMYCIN
ON URINARY
Day 1 0.56 0.95 0.73 0.69
2 & + +
0.52 0.65 0.66 0.44
1 CALCIUM
Day 2’ 0.84 1.91 0.63 0.76
a The data are expressed as milligrams per kilogram, b There were 10 dogs in each group. (- Neomycin was administered on Days 2 and 3. d Results are significant at a level ofp 5 0.025.
+ i & +
0.46 0.47d 0.49 0.53
EXCRETION
IN
Day 3c 0.89 1.96 0.37 1.48
? k & *
0.64 1.33d 0.17 0.59d
DOGP
Day 4 0.63 1.06 0.51 0.71
i 2 + *
0.28 0.58d 0.35 0.30
mean + SD.
In the first experiment, when 11 mg neomycin/kg was administered to the dogs, urinary calcium excretion was significantly increased on both treatment days (Days 2 and 3) and on one succeedingday (Day 4) (Table 1). Calcium excretion at the higher antibiotic dose did not follow the expected doseresponse relationship. Statistically significant urinary calcium excretion occurred only on Day 3 in this group. The consistent urinary excretion pattern in the group given 11 * Mycifradin
sulfate, The Upjohn
Co., Kalamazoo,
Michigan.
CA
EXCRETION
BY NEOMYCIN-TREATED
569
DOGS
mg/kg tended to verify the assumption that neomycin promotes urinary calcium excretion. Urine volume was not significantly affected at the 11-mg/kg antibiotic dose (Table 2). With 22 mg/kg as a dose, control urine volume progressively decreased.This made the resultsdifficult to interpret. TABLE 2 EFFECT
Dose(mg/kg)b
OF NEOMYCIN
ON URINE
Day 1
Day 2’
+ 8.1 24.1 f- 8.0 23.9 t 8.1
0 11
24.2 24.9 20.8 24.2
24.7
0 22
24.3
i
VOLUME
10.3
’ The data are expressed as milliliters per kilogram, b There were 10 dogs in each group. ’ Neomycin was administered on Days 2 and 3. d Results are significant at a level ofp I 0.005.
OF DOGP
Day 3’
t 8.0 k 9.8 f 9.3
Day 4 22.6 + 11.0 23.9 + 9.5 16.7 * 8.0 21.4 5 21.3d
24.5 & 9.1 24.8 + 1.6 15.8 + 4.2 29.8 k 23.1d
& 11.0 mean t SD.
TABLE 3 EFFECTOFNEOMYCINONSERUMCALCKJMOF DOGP Total Ca Dayb 1 2 3 4 5
Control 10.30 10.21 8.14 8.33 10.16
t + + k k
0.38 0.40 0.31 0.32 0.38
UnboundCa
Treated 9.77 9.62 8.14 8.61 9.84
31 0.49 k 0.41 f 0.33 k 0.35 f 0.44
Control 6.04 6.03 4.77 5.21 5.91
+ 0.60 3~ 0.60 i 0.41 k 0.45 k 0.60
BoundCa
Treated 6.29 5.76 4.91 5.50 5.98
+ + k 5 &
0.63 0.44 0.39 0.45 0.45
Control 4.15 k 4.18 rf3.38 k 3.12 & 4.25 &
0.81 0.88 0.66 0.51 0.89
Treated 3.56 3.76 3.23 3.11 3.82
k k t f +
0.60 0.59 0.55 0.68 0.65
” The data are expressed as milligrams per 100 milliliters, mean + SD. on Days 2 and 3. b Neomycin. 22 mg/kg. was administered
There were no differences in serum calcium concentrations between the control animals and those given 22 mg/kg of neomycin (Table 3). This finding contrasts with the results of bovine studies (Crawford et al., 1977) in which antibiotic administration produced hypocalcemia. This difference may be because4 hr elapsedbetween injection of the antibiotic and drawing of blood for calcium analysis in the present study, whereas in the bovine study blood sampleswere taken immediately after administration of the antibiotic. Perhaps mobilization of calcium into the blood stream from body stores had already compensated for the enhanced calcium excretion by the time the blood was drawn from the dogs. DISCUSSION The aminoglycoside seriesof antibiotics, including neomycin, has beenshown to bind calcium in a manner similar to that of ethylenediaminetetraacetic acid (EDTA) (Adams, 1975; Crawford and Bowen, 1971; Weinstein, 1965). It is also known that
570
CRAWFORD
AND
TESKE
EDTA promotes urinary calcium excretion by interfering with the active reabsorption of calcium in the proximal renal tubule (Simesen, 1970). Calcium complexed with EDTA is not ionized and thus is not susceptibleto the active reabsorption processes which are sensitive only to free ionized calcium. Passive reabsorption of calcium does occur in the loop of Henle and the collecting duct, but two thirds of the filtered load is reabsorbed in the proximal tubule (Koshanpour, 1976). Thus, it appears most probable that EDTA exerts its calcium excretory effect in that portion of the nephron. Neomycin enhancementof calcium excretion probably proceedsin a similar manner. The trend toward diuresis observed in the present experiment is perhaps related to increased urinary calcium. The tubular reabsorption of calcium is proportional to that of water (Simesen, 1970). Thus, prevention of tubular reabsorption of calcium results in the tubular retention of equimoiar amounts of water. This action is analogous to the action of those proprietary diuretic agentsthat act by obstructing sodium reabsorption in the proximal tubule (Levine, 1973). The findings of the present study tend to support the assumption that neomycin enhances the renal excretion of calcium (Koshanpour, 1976). On the basis of this finding, and becauseof the more immediate danger of neuromuscular blockade (Adams, 1975), calcium-compromised animals such as postpartum cows and bitches probably should not be treated with parenteral neomycin. ACKNOWLEDGMENTS
The authorsthank Patty Long andBruce Olcott for technicalsupport. The authors also acknowledgethe advice of Jerry Mitchell, Laboratory of Chemical Pharmacology,National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland. REFERENCES ADAMS, H. R. (1975). Acute adverse 987. CRAWFORD, L. M., AND BOWEN, J.
Am. J. Vet.Res.32, 357-359. CRAWFORD, L. M., CAMPBELL,
effects
M. (1971).
of antibiotics. Calcium
J.
Am. Vet. Med. Assoc. 166, 983-
binding
as a property
of kanamycin.
D. L., AND HARVEY, T. (1977). Hypocalcemic effect of aminoglycoside antibiotics in the dairy cow. Canad.J. Camp.Med. 41, 25 l-256. KOSHANPOUR, E. (1976). Renal Physiology: Principles and Functions. pp. 91, 244. W. B. Saunders, Philadelphia, Pa. LEVINE, R. R. (1973). Pharmacology: Drug Actions and Reactions.p. 285. Little, Brown, Boston, Mass. LOKEN, H. F., HAVEL, R. J., GORDON, G. S., AND WHITTINGTON, S. L. (1960). Ultracentrifugal analysis of protein-bound and free calcium in human serum. J. Biol. Chem.235, 3654-3658. SIMESEN, M. G. (1970). Calcium,inorganicphosphorus, and magnesium in healthand disease. In.Clinical Biochemistryof DomesticAnimals. (J. J. Kaneko and C. E. Cornelius, eds.), Vol. 1,2nd ed.,p. 318.AcademicPress,New York. WEINSTEIN, L. (1965). Chemotherapy of microbial diseases. In The PharmacologicalBasisof Therapeutics.(L. S. Goodman and (3. Gilman, eds.), 3rd ed., p. 123 1. Macmillan, New York.
