Several workers (Greenspan et al., 1950; Reinhardt and Li, 1951; Stack-Dunne and Young, 1951) have reported a lack of correlation between the eosinopenic, adrenal ascorbic acid lowering activity, and maintenance of adrenal weights with various adrenocorticotrophic preparations. Reinhardt et al. (1951) have critically evaluated assay methods for adrenocorticotrophic hormone (ACTH) and cautioned that differences in rate of absorption, variations in route of administration and dose-time-response relationships may influence the results obtained with different testing procedures. The study reported here was conducted under standardized conditions to determine whether the route of administration would influence the eosinopenic and adrenal ascorbic acid depleting properties of two ACTH preparations

Eversole, W. J.

Giere, Frederic A.

Name not available

Adrenal Ascorbic Acid Depletion Without Eosinopenia in Hypophysectomized Rats Injected lntrasplenically with Corticotrophin

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 63 Annual Issue Article 78 1956 Adrenal Ascorbic Acid Depletion Without Eosinopenia in Hypophysectomized Rats Injected lntrasplenically with Corticotrophin Frederic A. Giere Luther College W. J. Eversole University of New Mexico Let us know how access to this document benefits you Copyright ©1956 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Giere, Frederic A. and Eversole, W. J. (1956) "Adrenal Ascorbic Acid Depletion Without Eosinopenia in Hypophysectomized Rats Injected lntrasplenically with Corticotrophin," Proceedings of the Iowa Academy of Science, 63(1), 693-696. Available at: https://scholarworks.uni.edu/pias/vol63/iss1/78 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. Adrenal Ascorbic Acid Depletion Without Eosinopenia in Hypophysectomized Rats Injected lntrasplenically with Corticotrophin By FREDERIC A. GIERE AND \V. J. EVERSOLE Several workers (Greenspan et al., 1950; Reinhardt and Li, 1951; Stack-Dunne and Young, 1951) have reported a lack of correlation between the eosinopenic, adrenal ascorbic acid lower-ing activity, and maintenance of adrenal weights with various adrenocorticotrophic preparations. Reinhardt et al. ( 1951) have critically evaluated assay methods for adrenocorticotrophic hor-mone (ACTH) and cautioned that differences in rate of absorp-tion, variations in route of administration and dose-time-response relationships may influence the results obtained with different testing procedures. The study reported here was conducted under standardized conditions to determine whether the route of ad-ministration would influence the eosinopenic and adrenal ascorbic acid depleting properties of two ACTH preparations. Hypophysectomized male Sprague-Dawley rats weighing ap-proximately 170 grams were used in groups of eights, 24 to 48 hours post-hypophysectomy. Animals were injected with ACTH either intrasplenically or subcutaneously. The ACTH was dissolved in 0.9% sodium chloride; the volume of injected fluid was 0.2 ml. in all cases. Those animals that were injected with ACTH subcu-taneously received saline intrasplcnically and those injected with ACTH intrasplenically received saline subcutaneously. This pro-cedure controlled the possibility of changes in eosinophil counts due to mechanical flushing of the spleen. The procedure employed in obtaining the adrenals for ascorbic acid determinations was that of Sayers, Sayers, and Woodbury (1948). Each rat was anesthetized with pentobarbital sodium, one adrenal was removed for ascorbic acid determination and the spleen was exposed for injection. After injection the spleen was returned to the peritoneal cavity and the incision was closed. Additional hypophysectomized animals re-ceived injections of ACTH via the femoral vein. In all cases the left adrenal was removed just prior to hormone injection and the right adrenal was removed one hour after injection. Ascorbic acid concentration of the adrena.ls was determined by the method of Roe and Kuether ( 1943). A sample of tail blood was taken simul-taneously with removal of each adrenal. The blood was diluted with phloxine-propylene glycol diluting fluid and eosinophil counts made according to the procedure recommended by Henneman, Wexler, and Westenhaver ( 1949) . The criteria used in determining 693 1Giere and Eversole: Adrenal Ascorbic Acid Depletion Without Eosinopenia in HypophysecPublished by UNI ScholarWorks, 1956694 IOWA ACADEMY OF SCIENCE [Vol. 63 the effectiwness of the ACTH preparations were changes m cosinophil counts and adrenal ascorbic acid concentrations. Table I summarizes the results. There was depiction of adrenal ascorbic acid with all quantities of both ACTH preparations re-gardless of the site of injection. In experiments with sufficient numbers of cases for statistical analyses the concentration of adrenal ascorbic acid in the "treated" right adrenal was significantly lower than in the control left adrenal (P<0.01). Subcutaneous or intra-venous administration of ACTH, exception four micrograms given intravenously, resulted in a fall in eosinophils; when four micro-grams were given intravenously the fall in eosinophils was negligible. Intrasplenic injection of ACTH at all dosage levels resulted in no significant changes in eosinophil counts. Cheng, Sayers, Goodman and Swinyard ( 1949) have reported adrenal ascorbic acid depletion after histamine injection in rats bearing aclenohypophysis transplants to the spleen. Our data supports their conclusion that absorption in the spleen or passage through the liver does not alter the adrenal ascorbic acid depleting properties of ACTH. HO\vever, our experiments do indicate that the eosinopenic activity of ACTH is modified by passage through the liver and/ or spleen. Reinhardt, Hungerford, and Li ( 1951) have found that there is no obvious close correlation between the eosinopcnic activity of either ACTH protein or peptide and the degree of adrenal ascorbic acid lowering activity. In our experi-ments ACTH, when injected subcutaneously, consistantly caused a drop in eosinophils but did not cause a drop when injected intra-splenically. One possible explanation for such findings is that the spleen and/or the liver modified the ACTH preparations used here in such a fashion as to allow an adrenal ascorbic acid depleting factor to escape into the general circulation but retained or in-activated a fraction involved in lowering the eosinophils. Such an interpretation would be compatible with that of Hungerford, et al., (1952) who, on the basis of their findings, suggested that an eosin-openic factor may be a separate component of ACTH different from an adrenal ascorbic acid depleting factor. Alternatively, it is possible that, in our experiments, a time interval greater than one hour would be necessary for intrasplenically administered ACTH to cause an eosinopenia. Our finding that it requires smaller amounts of intravenously administered ACTH to cause a lowering of the adrenal ascorbic acid than it does to cause an eosinopenia may also have a bearing on the results obtained after intrasplenic ad-ministration. It may be that the spleen and liver acted as a filtering device and allowed only that amount of ACTH to reach the gen-eral circulation that would affect adrenal ascorbic acid but not eosinophils. 2Proceedings of the Iowa Academy of Science, Vol. 63 [1956], No. 1, Art. 78https://scholarworks.uni.edu/pias/vol63/iss1/78Table 1. Effects of route of administration on activity of ACTH. Values are given as mean ± S. E. (IS=Intrasplenic; IV=Intravenous; SQ=Subcutaneous) Adrenal Ascorbic Acid Eosinophils /cmm Method of No. expressed as mg % ~~ Dosage Admini- Rats Change stration Lt. Adr. Rt. Adr. Before After Before ACTH After ACTH ACTH ACTH Lot 71-50 R 9 mg IS 6 440±21.8 245±17.2 -44 241±30.0 280±39.1 9 mg SQ 7 397± 7.2 226± 10.4 -43 317±33.9 221±49.7 Lot 128-105R 8 mg IS 4 431±14.6 271± 8.9 - 37 220±43.1 247±47.4 4 mg IS 1 300 163 -45 133 111 2 mg IS 2 328± 4.0 185± 5.0 -44 122±11.0 105±17.0 1 mg IS 4 366± 10.2 227±30.7 - 38 151±12.2 156±22.4 16 gamma IS 5 391± 9.7 237±10.5 - 43 349± 17.9 341±24.2 1 mg IV 3 404± 7.0 231±17.4 - 39 117::±:13.2 92±13.2 16 gamma IV 7 345±19.9 211±10.1 - 39 183±41.1 126±29.5 4 gamma IV 4 426::':: 13.8 280± 7.6 - 34 231±30.8 222±35.1 % Change +16 -30 +12 - 16 -14 - 3 - 2 - 2 l - 31 - 4 -<O (J1 ~ >-en 0 0 ~ t:C -0 >-0 -t:i t:i t"l 'ti t"' t"l >-l -0 z a> <O (J1 3Giere and Eversole: Adrenal Ascorbic Acid Depletion Without Eosinopenia in HypophysecPublished by UNI ScholarWorks, 1956696 IOWA ACADEMY OF SCIENCE (Vol. 63 This investigation was supported in part by a research grant ( A-202) from the National Institute of Arthritis and Metabolic Diseases, of the National Institutes of Health, Public Health Service. The ACTH preparations were supplied by Dr. Irby Bunding of the Armour Laboratories. The activity of the ACTH preparations, as tested by Armour and compared to their LA-1-A standard was as follows: Lot 71-50 was 20 per cent of the standard; Lot 128-105 was 1.6 times the standard. Literature Cited Cheng, C. P., Sayers, G., Goodman, L. S. and Swinyard, C. A. 1949. Discharge of Adrenocorticotrophic Hormone from transplanted Pitu-itary tissue. Am. J. Physiol. 159:426-432. Greenspan, F. S., Li, C. H., Simpson, M. E. and Evans, H. M. 1950. Hormone Assay, ed. by Emmons, C. W., Academic Press, Inc. N. Y. Henneman, P., Wexler, H. and Westenhaver, M. J. 1949. A comparison of Eosin-Acetone and Phloxine-Propylene Glycol dilutents in eosino-phil counts. J. Lab. and Clin. Med. 34: J 017. Hungerford, G. F., Reinhardt, W. 0., and Li, C. H. 1952. Lack of cor· relation of eosinopcnic and adrenal ascorbic acid-depleting activities of various adrenocorticotrophic preparations. Proc. Soc. Exptl. Biol. & Med. 81 :'.120-323. Reinhardt, W. 0., and Li, C. H. 1951. Apparent discrepancies in evaluation of adrenocorticotrophic hormone (ACTH) activity by two ass;:iv methods. Proc. Soc. Exptl. Biol. & Med. 77:229-23 J. Reinhardt, W. 0., Hungerford, P. F., and Li, C. H. 1951. Eosinopenic r<'sponse of mice to ACTH protein and peptide. Fed. Proc. 10:109. Reinhardt, W. 0., Geschwind, I. I. and Li, C. H. 1951. On the evidence suggesting a multiplicity of adrcnocorticotrophic hormones. Acta. Endocrinol. 8:393-424. Roe, J. H. and Kuether, C. A. J 943. The determination of Ascorbic Acid in whok blood and urine through the 2, 4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem. 147:399-497. Sayers, M. A., Sayers, G. and L. A. Woodbury. 1948. The assay of adrenocorticotrophic hormone by the adrenal ascorbic acid-depleting method. Endocrinol. 42:379-393. Stack-Dunne, M. and Young, F. G. 1951. The properties of ACTH. J. Endocrinol. 7 :lxvi-lxix. DEPARTMENTS OF BIOLOGY LUTHER COLLEGE DECORAH, IowA AND UNIVERSITY OF NEW MEXICO ALBUQUERQUE, NEW MEXICO 4Proceedings of the Iowa Academy of Science, Vol. 63 [1956], No. 1, Art. 78https://scholarworks.uni.edu/pias/vol63/iss1/78

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Adrenal Ascorbic Acid Depletion Without Eosinopenia in Hypophysectomized Rats Injected lntrasplenically with Corticotrophin

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