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Effect of Carbonic Anhydrase Inhibition on Blood Acid-Base Balance in the Chicken Embryo
Effect of Carbonic Anhydrase Inhibition on Blood Acid-Base Balance in the Chicken Embryo GEOFFREY F. BIRCHARD 1 and CRAIG P. BLACK Department of Biology, University of Toledo, Toledo, Ohio 43606 (Received for publication October 24, 1984) ABSTRACT The role of carbonic anhydrase in chicken embryo blood acid-base balance was examined using the carbonic anhydrase inhibitor acetazolamide during the second half of in.cubation. Day 13, 16, and 17 embryos of treated eggs had significant increases in blood P Q Q and decreases in blood pH when compared with controls. (Key words: carbonic anhydrase, acid-base balance, chicken embryo) 1986 Poultry Science 65:1811-1813 INTRODUCTION
MATERIALS AND METHODS
Fertile eggs (White Leghorn) were obtained from a local supplier and placed in a Humidaire Model 14 Gooser incubator with wet and dry bulb temperatures of 29.7 and 37.6 C, respectively. Acetazolamide (Sigma) was administered as one dose (3 mg/egg, equal to approximately 100 mg/kg in the largest embryos) on either Day 12, 15, or 16. A small hole was drilled through the shell at the equator and the drug (suspended in .9% saline, 3 mg/.l ml) was injected with a 25-guage needle into the al-
1 Present address for correspondence: Department of Biology, George Mason University, 4400 University Dr., Fairfax, VA 22030.
bumin just below the chorioallantoic membrane. Sham controls were injected with saline (.9%) and normal controls were untreated. Holes were sealed with a mixture of epoxy and ground egg shells. Eggs were returned to the incubator to hatch or be sampled 24 hr later. Blood samples were drawn anaerobically from the chorioallantoic artery (Day 13) or vein (Days 16 and 17) using the methods of Tazawa et al. (1971). Blood C 0 2 -buffering capacity (Alog P c o 2 / A p H ) was determined for Day 16 embryos by tonometry. Blood was tonometered for 12 min in an Astrup microtonometer containing 30% 0 2 with 3, 5.5, or 8% C 0 2 and the balance N 2 . Blood gases and pH were measured with a Radiometer BMS-3 MK11 Blood microsystem in conjuction with a PHM-72 MK11 Digital Acid-Base Analyzer at 37 C. Bicarbonate was calculated with the Henderson-Hasselbalch equation using a pK value of 6.09 (Helbacka et al, 1964) and an a of .032 (Severinghaus et al, 1956). Differences between means were assessed by a Student's t-test. A p value of less than .05 was considered significant. RESULTS AND DISCUSSION
Hatchability of Day 15 treated eggs was 92% (12/13) in untreated controls, 93% (13/14) in sham controls, and 93% (13/14) in acetazolamide-injected eggs. In contrast to previous studies (Zwilling and DeBell, 1950; Landauer and Wakasugi, 1967) no teratogenic effects were evident in newly hatched chicks of acetazolamide-treated eggs. The lack of a teratogenic effect with actazolamide treatment may be due to the time of treatment or the small number of eggs per treatment.
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Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015
The enzyme carbonic anhydrase catylyzes the reaction C 0 2 + H 2 0 = H 2 C 0 3 . This enzyme is involved in blood C 0 2 transport and many ionic transport processes in biological systems (Bundy, 1977). Although it has long been known that embryonic chicken tissues contain carbonic anhydrase, little work exists on its physiological role (Clark, 1951; Gay et al, 1981). This investigation was designed to examine the role of carbonic anhydrase in blood acid-base balance during the second half of incubation by using the drug, acetazolamide, to inhibit enzyme activity. The last half of incubation was chosen as blood carbonic anhydrase does not appear until Day 12 (Clark, 1951) and C 0 2 production and the need for acid-base regulation is greatest during this time (Dawes, 1975).
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BIRCHARD AND BLACK TABLE 1. Blood P^Q , PQ , and pH for acetazolamide-injected after injection (mean ± SE) N
pH
and control eggs 24 br
PO2
PCO,
(HCO3-) (meq/liter)
Hg) •
Day 13 Control Acetazolamide injected
6 6
7.449 + .022 7.363 + .020**
23.2 ± .9 33.1 + 1.6*
34.1 ± 1.1 26.3 ± 4.0
16.9 ± .7 20.1 ± 1.5
Day 16 Control Acetazolamide injected
7 10
7.484 ± .011 7.405 + .008**
32.1 ± 1.0 42.8 ± 1.5**
60.6 ± 3.5 54.1 ± 3.4
25.5 ± 1.1 28.2 ± 1.0
Day 17 Control Acetazolamide injected
10 11
7.474 + .011 7.415 ± .008**
35.1 ± 1.4 41.9 ± 1.4**
52.8 ± 2.6 60.8 + 2.9
27.1 ± .7 28.3 ± .7
**P<.001.
Blood gas and pH results are shown in Table 1. Data for sham and untreated controls were not different and were combined. Day 16 and 17 control values agree well with previously published results using this technique (Tazawa et al, 1971). In Day 13, control embryos' blood pH and P Q , values were higher and P C 0 2 values lower than those reported by Tazawa et al. (1971). Acetazolamide treatment significantly increased blood P c 0 2 an<^ ^ecreased pH. The single dose used here most likely gives the maximal blood carbonic anhydrase inhibition obtainable with this technique, as preliminary experiments showed multiple treatments (Days 11, 13, 15, and 16) yield similar blood pH and gas values (pH = 7.367 ± .011, P C 0 2 = 40.0 ± 1.5, P o 2 = 61.8 ± 1.9; N = 6). On the basis of the in vitro C 0 2 buffering capacity (—1.14), the decreased pH appears to be the result of the increased P Q O 2 • The changes in blood gases and pH seen were similar to those described in adult birds and fetal mammals (Longo et al, 1974; Powell et al, 1978). The results of this study clearly indicate a role for carbonic anhydrase in normal avian embryonic blood acid-base balance. Whether the elevated P c 0 2 S e e n n e r e a r e the sole result of blood carbonic anhydrase, or, in addition, choriallantoic membrane enzyme (Reider et al, 1980; Narbaitz et al, 1981) facilitating C 0 2 flux as in the mammalian lung (Enns, 1967; Effros et al, 1978) cannot be determined. That hatchability was apparently unaffected by carbonic anhydrase inhibition, leads to the
question of the importance of this enzyme during the last half of incubation. It may indicate that the uncatalyzed rate of C 0 2 hydration is sufficient in ovo and that various processes (e.g., calcium uptake from the shell) may not be as dependent on this enzyme as previous experiments indicate (Tuan and Zrike, 1978). ACKNOWLEDGMENTS
Funds in support of this research were provided by the Faculty Research and Development Fund of the University of Toledo Graduate School awarded to CPB. We thank F. Bennett for helpful criticism on an earlier draft of this paper.
REFERENCES Bundy, H., 1977. Carbonic anhydrase. Comp. Biochem. Physiol. 5 7 B : l - 7 . Clark, A. M., 1951. Carbonic anhydrase activity during embryonic development. J. Exp. Biol. 28: 332-343. Dawes, C. M., 1975. Acid-base relationships with the avian egg. Biol. Rev. 5 0 : 3 5 1 - 3 7 1 . Effros, R. M., R. S. Chang, and P. S. Silverman, 1978. Acceleration of plasma bicarbonate conversion to carbon dioxide by pulmonary carbonic anhydrase. Science 199:427-429. Enns, T., 1967. Facilitation by carbonic anhydrase of carbon dioxide transport. Science 155:44—47. Gay, C. V., H. Schraer, D. J. Sharkey, and E. Rieder, 1981. Carbonic anhydrase in developing heart, blood and chorioallantoic membrane. Comp. Biochem. Physiol. 70A:173-177. Helbacka, N. V., J. L. Casterline, C. J. Smith, and C. S. Shaffner, 1964. Investigation of plasma carbonic
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015
*P<.01.
RESEARCH NOTE acid pk of the chicken. Poultry Sci. 43:138—144. Landauer, W., and N. Wakasugi, 1967. Problems of acetazolamide and n-ethylnicotinamide as teratogens. J. Exp. Zool. 164:499-516. Longo, L. D., M. Delivoria-Papadopoulos, and R. E. Forster II, 1974. Placental C 0 2 transfer after fetal carbonic anhydrase inhibition. Am. J. Physiol. 226:703-710. Narbaitz, R., S. Kacew, and L. Sitwell, 1981. Carbonic anhydrase activity in the chick embryo chroioallantois: regional distribution and vitamin Dl regulation. J. Embryol. Exp. Morphol. 65: 127-137. Powell, F. L., M. R. Fedde, Fr. K. Gratz, and P. Scheid, 1978. Ventilatory response to C 0 2 in birds. I. Measurements in the unanesthetized duck. Respir. Physiol. 35:349-359. Reider, E., C. V. Gay, and H. Schraer, 1980. Au-
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toradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane. Anat. Embryol. 159:17-31. Severinghaus, J. W., M. Stupfel, and A. F. Bradley, 1956. Accuracy of blood pH and pC0 2 determinations. J. Appl. Physiol. 9:189—196. Tazawa, H., T. Mikami, and C. Yoshimoto, 1971. Respiratory properties of chicken embryonic blood during development. Respir. Physiol. 13:160-170. Tuan, R. S., and J. Zrike, 1978. Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane. Biochem. J. 176:67-74. Zwilling, E., and J. T. DeBell, 1950. Micromelia and growth retardation as independent effects of sulfanamide in chick embryos. J. Exp. Zool. 115:59-81. Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015
MATERIALS AND METHODS
Fertile eggs (White Leghorn) were obtained from a local supplier and placed in a Humidaire Model 14 Gooser incubator with wet and dry bulb temperatures of 29.7 and 37.6 C, respectively. Acetazolamide (Sigma) was administered as one dose (3 mg/egg, equal to approximately 100 mg/kg in the largest embryos) on either Day 12, 15, or 16. A small hole was drilled through the shell at the equator and the drug (suspended in .9% saline, 3 mg/.l ml) was injected with a 25-guage needle into the al-
1 Present address for correspondence: Department of Biology, George Mason University, 4400 University Dr., Fairfax, VA 22030.
bumin just below the chorioallantoic membrane. Sham controls were injected with saline (.9%) and normal controls were untreated. Holes were sealed with a mixture of epoxy and ground egg shells. Eggs were returned to the incubator to hatch or be sampled 24 hr later. Blood samples were drawn anaerobically from the chorioallantoic artery (Day 13) or vein (Days 16 and 17) using the methods of Tazawa et al. (1971). Blood C 0 2 -buffering capacity (Alog P c o 2 / A p H ) was determined for Day 16 embryos by tonometry. Blood was tonometered for 12 min in an Astrup microtonometer containing 30% 0 2 with 3, 5.5, or 8% C 0 2 and the balance N 2 . Blood gases and pH were measured with a Radiometer BMS-3 MK11 Blood microsystem in conjuction with a PHM-72 MK11 Digital Acid-Base Analyzer at 37 C. Bicarbonate was calculated with the Henderson-Hasselbalch equation using a pK value of 6.09 (Helbacka et al, 1964) and an a of .032 (Severinghaus et al, 1956). Differences between means were assessed by a Student's t-test. A p value of less than .05 was considered significant. RESULTS AND DISCUSSION
Hatchability of Day 15 treated eggs was 92% (12/13) in untreated controls, 93% (13/14) in sham controls, and 93% (13/14) in acetazolamide-injected eggs. In contrast to previous studies (Zwilling and DeBell, 1950; Landauer and Wakasugi, 1967) no teratogenic effects were evident in newly hatched chicks of acetazolamide-treated eggs. The lack of a teratogenic effect with actazolamide treatment may be due to the time of treatment or the small number of eggs per treatment.
1811
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015
The enzyme carbonic anhydrase catylyzes the reaction C 0 2 + H 2 0 = H 2 C 0 3 . This enzyme is involved in blood C 0 2 transport and many ionic transport processes in biological systems (Bundy, 1977). Although it has long been known that embryonic chicken tissues contain carbonic anhydrase, little work exists on its physiological role (Clark, 1951; Gay et al, 1981). This investigation was designed to examine the role of carbonic anhydrase in blood acid-base balance during the second half of incubation by using the drug, acetazolamide, to inhibit enzyme activity. The last half of incubation was chosen as blood carbonic anhydrase does not appear until Day 12 (Clark, 1951) and C 0 2 production and the need for acid-base regulation is greatest during this time (Dawes, 1975).
1812
BIRCHARD AND BLACK TABLE 1. Blood P^Q , PQ , and pH for acetazolamide-injected after injection (mean ± SE) N
pH
and control eggs 24 br
PO2
PCO,
(HCO3-) (meq/liter)
Hg) •
Day 13 Control Acetazolamide injected
6 6
7.449 + .022 7.363 + .020**
23.2 ± .9 33.1 + 1.6*
34.1 ± 1.1 26.3 ± 4.0
16.9 ± .7 20.1 ± 1.5
Day 16 Control Acetazolamide injected
7 10
7.484 ± .011 7.405 + .008**
32.1 ± 1.0 42.8 ± 1.5**
60.6 ± 3.5 54.1 ± 3.4
25.5 ± 1.1 28.2 ± 1.0
Day 17 Control Acetazolamide injected
10 11
7.474 + .011 7.415 ± .008**
35.1 ± 1.4 41.9 ± 1.4**
52.8 ± 2.6 60.8 + 2.9
27.1 ± .7 28.3 ± .7
**P<.001.
Blood gas and pH results are shown in Table 1. Data for sham and untreated controls were not different and were combined. Day 16 and 17 control values agree well with previously published results using this technique (Tazawa et al, 1971). In Day 13, control embryos' blood pH and P Q , values were higher and P C 0 2 values lower than those reported by Tazawa et al. (1971). Acetazolamide treatment significantly increased blood P c 0 2 an<^ ^ecreased pH. The single dose used here most likely gives the maximal blood carbonic anhydrase inhibition obtainable with this technique, as preliminary experiments showed multiple treatments (Days 11, 13, 15, and 16) yield similar blood pH and gas values (pH = 7.367 ± .011, P C 0 2 = 40.0 ± 1.5, P o 2 = 61.8 ± 1.9; N = 6). On the basis of the in vitro C 0 2 buffering capacity (—1.14), the decreased pH appears to be the result of the increased P Q O 2 • The changes in blood gases and pH seen were similar to those described in adult birds and fetal mammals (Longo et al, 1974; Powell et al, 1978). The results of this study clearly indicate a role for carbonic anhydrase in normal avian embryonic blood acid-base balance. Whether the elevated P c 0 2 S e e n n e r e a r e the sole result of blood carbonic anhydrase, or, in addition, choriallantoic membrane enzyme (Reider et al, 1980; Narbaitz et al, 1981) facilitating C 0 2 flux as in the mammalian lung (Enns, 1967; Effros et al, 1978) cannot be determined. That hatchability was apparently unaffected by carbonic anhydrase inhibition, leads to the
question of the importance of this enzyme during the last half of incubation. It may indicate that the uncatalyzed rate of C 0 2 hydration is sufficient in ovo and that various processes (e.g., calcium uptake from the shell) may not be as dependent on this enzyme as previous experiments indicate (Tuan and Zrike, 1978). ACKNOWLEDGMENTS
Funds in support of this research were provided by the Faculty Research and Development Fund of the University of Toledo Graduate School awarded to CPB. We thank F. Bennett for helpful criticism on an earlier draft of this paper.
REFERENCES Bundy, H., 1977. Carbonic anhydrase. Comp. Biochem. Physiol. 5 7 B : l - 7 . Clark, A. M., 1951. Carbonic anhydrase activity during embryonic development. J. Exp. Biol. 28: 332-343. Dawes, C. M., 1975. Acid-base relationships with the avian egg. Biol. Rev. 5 0 : 3 5 1 - 3 7 1 . Effros, R. M., R. S. Chang, and P. S. Silverman, 1978. Acceleration of plasma bicarbonate conversion to carbon dioxide by pulmonary carbonic anhydrase. Science 199:427-429. Enns, T., 1967. Facilitation by carbonic anhydrase of carbon dioxide transport. Science 155:44—47. Gay, C. V., H. Schraer, D. J. Sharkey, and E. Rieder, 1981. Carbonic anhydrase in developing heart, blood and chorioallantoic membrane. Comp. Biochem. Physiol. 70A:173-177. Helbacka, N. V., J. L. Casterline, C. J. Smith, and C. S. Shaffner, 1964. Investigation of plasma carbonic
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015
*P<.01.
RESEARCH NOTE acid pk of the chicken. Poultry Sci. 43:138—144. Landauer, W., and N. Wakasugi, 1967. Problems of acetazolamide and n-ethylnicotinamide as teratogens. J. Exp. Zool. 164:499-516. Longo, L. D., M. Delivoria-Papadopoulos, and R. E. Forster II, 1974. Placental C 0 2 transfer after fetal carbonic anhydrase inhibition. Am. J. Physiol. 226:703-710. Narbaitz, R., S. Kacew, and L. Sitwell, 1981. Carbonic anhydrase activity in the chick embryo chroioallantois: regional distribution and vitamin Dl regulation. J. Embryol. Exp. Morphol. 65: 127-137. Powell, F. L., M. R. Fedde, Fr. K. Gratz, and P. Scheid, 1978. Ventilatory response to C 0 2 in birds. I. Measurements in the unanesthetized duck. Respir. Physiol. 35:349-359. Reider, E., C. V. Gay, and H. Schraer, 1980. Au-
1813
toradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane. Anat. Embryol. 159:17-31. Severinghaus, J. W., M. Stupfel, and A. F. Bradley, 1956. Accuracy of blood pH and pC0 2 determinations. J. Appl. Physiol. 9:189—196. Tazawa, H., T. Mikami, and C. Yoshimoto, 1971. Respiratory properties of chicken embryonic blood during development. Respir. Physiol. 13:160-170. Tuan, R. S., and J. Zrike, 1978. Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane. Biochem. J. 176:67-74. Zwilling, E., and J. T. DeBell, 1950. Micromelia and growth retardation as independent effects of sulfanamide in chick embryos. J. Exp. Zool. 115:59-81. Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 23, 2015