Effect of Corticotropin on Ascorbic Acid Metabolism
By MAHESH G. NATHANI AND M. C. NATH Corticotropin (40 I.U./kg body weight) was injected i.p. to intact and adrenalectomized rats. One hour after injection, the rats were sacrificed and their ascorbic acid, dehydroascorbic acid, and diketogulonic acid levels were determined in liver, while only the total ascorbic acid was determined in blood. Activities of ascorbic acid synthesizing enzymes, u-glucurono-Mactone hydrolase, L-gulono-y-lactone hydrolase, TPN-L-hexonate dehydrogenase and L-gulono-ylactoue oxidase were estimated in liver. The activities of ascorbic acid degrading enzymes, dehydroascorbatase and 2,3 diketoaldonate decarboxylase were studied in liver and kidney. In corticotropin-
administered normal rats there was slight increase in ascorbic acid and dehydroascorbic acid contents of liver as compared to control (normal rats). There was a decrease in the diketogulonic acid content of liver. Corticotropin had no appreciable effect on the activities of any of the ascorbic acid synthesizing enzymes in normal rats. Under similar conditions, there was an appreciable decrease in the activities of dehydroascorbatase and 2,3 diketoaldonate decarboxylase both in liver and kidney. It has been suggested that corticotropin does not work directly but indirectly through the adrenocortical axis since it had no effect in adrenalectomized rats.
T HAS BEEN SHOWN by various workers that adrenal cortex plays an important role in ascorbic acid metabolism. Recently it has been reported from this laboratory that adrenalectomy decreases ascorbic acid and dehydroascorbic acid but increases diketogulonic acid contents of 1iver.l These changes were mostly due to increased ascorbic acid catabolism in adrenalectomized rats. Adrenalectomy, however, caused decrease in the activities of some ascorbic acid synthesizing enzymes such as o-glucurono-S-lactone hydrolase and Lgulono-y-lactone oxidase in rat liver. It has shown by Allison2 that the stimulation of pituitary-adrenal axis increases the blood ascorbic acid. Corticotropin administration has been reported to increase the ascorbic acid content of liver and blood,3,4 but no attempts have so far been made to study the effect of corticotropin on ascorbic acid synthesizing and degrading enzymes. (Such studies seem to be essential to evaluate the exact role played by corticotropin in increasing the ascorbic acid content of liver and blood.) It was thought worthwhile, therefore, to examine the effect From the University Department of Biochemistry, Mahatma Gandhi Mat-g, Nagpur, India. Received for publication August 20, 1971. Supported in part by the Indian Council of Medical Research. MAHESH G. NATHANI, MSc.: Research Fellow, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. M. C. NATH, D.&L, F.N.I., F.R.I.C.: Professor Under University Grants Commission Retired Teacher’s Scheme, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. METABOLISM, VOL. 21, No. 2 (FEBRUARY), 1972
NATHAN1 AND NATH
of corticotopin on the contents of ascorbic acid, dehydroascorbic acid, and diketogulonic acid in liver and blood and on the various enzymes that bring about the metabolism of ascorbic acid in rat liver and kidney. MATERIALS
n-Glucuronic acid lactone and L-gluono-y-lactone were obtained from Sigma CO., St. Louis, MO. NADPH was purchased from the British Drug House, Ltd., Poole, England. Corticotropin (source, pig) was obtained from Fredriksberg Chemical Lab., Ltd., Denmark. Bovine serum albumin and sodium glucuronate were purchased from Nutritional Biowas prepared by the method of chemicals Corp., Cleveland, Ohio. 2,s Diketogulonate Kagawa.6 All other chemicals were of analytical grade. Male albino rats (10&125-g body weight) were distributed into five groups of six each and were maintained on stock laboratory diet. s Rats of groups 1, 2, and 3 were normal rats. Rats from Groups 4 and 5 were adrenalectomized bilaterally and were maintained on the same stock laboratory diet. Water was replaced by 0.9% (w/v) saline. After 8 days of adrenalectomy they were used for experiments. The rats in Group 1 served as uninjected controls. Group 3 and 5 rats were injected with 40 I.D. of corticotropin/kg body weight i.p. Rats in Groups 2 and 4 were similarly treated with isotonic saline. Saline (1.0 ml, 0.9%, w/v) was injected in control groups 2 and 4) to obviate the condition of stress as a result of injection. One hour after injection, the rats were killed by decapitation and blood was collected from the neck in oxalated tubes for the estimation of total ascorbic acid (ascorbic acid, dehydroascorbic acid, and diketogulonic acid). Liver and kidneys were removed and rinsed in ice-cold water and blotted dry. Part of the tissue was homogenized in 9 volumes of isotonic sucrose. The homogenates were centrifuged at 10,000 g for 20 min to obtain tissue extract free of heavy particles as the supernate. The supernate thus obtained was again centrifuged at 100,000 g for 1 hr to yield microsomes and soluble fractions. Another portion of the tissue was homogenized in 4 volumes of isotonic sucrose and the homogenates obtained were used for the assay of the complete system. Different estimations were carried out as follows. Ascorbic acid, dehydroascorbic acid, and diketogulonic acid were estimated in liver and blood by the method of Roe et al.7 n-Glucurono-&lactone hydrolase (EC 22.214.171.124) activity was estimated by the modified method of Salomon et al.8 The incubation mixture consisted of microsomes equivalent to 8.75 mg liver, 0.04 M NaHCO,, 0.04 M n-glucurono lactone (final volume, 2 ml), pH 7.6, 2S’, 95% N,-5% COa. Activity was determined manometrically by CO, evolution for 1 hr. Specific activity was defined as microliters of CO, evolved per milligram of protein at 30-min intervals under assay conditions. r_-Gulono-y-lactone hydrolase (EC 126.96.36.199) activity enzyme was estimated by the modified method of Salomon et al.8 The incubation mixture consisted of soluble fraction equivalent to 3.75 mg liver, 0.04 M NaHCO,, 0.07 M L-gulono-y-lactone (final volume, 1 by CO, ml), pH 7.6, 25’, 95% N,-5% CO,. Activity was determined manometrically evolution for 1 hr. Specific activity was defined as microliters of CO, evolved per milligram of protein at 30-min intervals under assay conditions. TPN-L-hexonate dehydrogenase (L-gulono-y-lactone: NADP oxidoreductase, EC 188.8.131.52) activity was estimated by the modified method of Salomon et al.8 The incubation mixture consisted of soluble fraction equivalent to 20 mg liver, 0.003 M D-glucuronolactone, 0.01 M nicotinamide, 0.001 M TPNH, 0.01 M KCI, 0.05 M Tris (hydroxymethyl) aminomethaneHCl buffer, final volume: 3 ml, pH 7.6. The change in the optical density was measured at 340 rn~~for 20 min. The specific activity was defined as change in optical density per milligram of protein at lo-min intervals X 10.3 L-Gulono-y-lactone oxidase (L-gulono-y-lactone: oxido-reductase, EC 184.108.40.206) activity was estimated by the modified method of Salomon et al.9 The incubation mixture consisted of microsomes suspended in 0.05 M Tris (hydroxymethyl) aminomethane-HCl buffer, pH 7.6, equivalent to 120 mg liver, 0.00042 M L-gulono-y-lactone, 0.02 M glutathione adjusted to pH 7.6 with NaHCO, (all concentrations final), total volume: 2.0 ml: and
CORTICOTROPIN AND ASCORBIC ACID
Table l.-Effect of Coticotropin on Ascorbic Acid, Debydroascorbic Acid, and Diketogulonic Acid; Contents of Liver and Blood in Normal and Adrenalectomized Bats Liver (mg/lOO g Fresh Liver) Groups
Normal Rats 1 2 Saline 3 Corticotropin Adrenalectomized Rats 4 Saline 5 Corticotropin Values *p < t p < $p <
21.36 22.17 27.85
+ 0.93 + 1.27 r 1.82*
17.735 +- 1.10 17.162 r 1.77
Blood (mg/lOO ml Blood)
3.17 r 0.12 3.42 I!I 0.08 3.79 Z!I0.21t
1.51 1.73 0.81
1.39 t 0.15 1.57 + 0.17
3.310 f 0.21 3.640 -I- 0.15
+ 0.09 2 0.06 ? 0.19*
Total Ascorbic Acid
1.130 r 0.025 1.121 t 0.020 1.358 I!I 0.028$ 0.741 I? 0.040 0.820 -r- 0.060
are expressed as mean f SEM of six rats. 0.02 as compared to Group 1 using Student’s t test. 0.05 as compared to Group 1 using Student’s t test. 0.001 as compared to Group 1 using Student’s t test.
incubated for 1 hr under 0, at 25O. After 1 hr, 1 ml of 10% trichloroacetic acid (TCA) was added to stop the reaction and the activity was measured by ascorbate production. The specific activity has been defined as @ales of ascorbic acid formed per milligram of protein per hour under assay conditions. The complete system was assayed by the modified method of Salomon et a1.s The incubation mixture consisted of liver extract equivalent to 120 mg liver, 0.05 M Tris (hydroxy-methyl) aminomethane-HCI buffer, 0.002 M sodium-D-glucuronate adjusted to pH 7.6, 0.0008 M TPNH, 0.01 M nicotinamide, 0.02 M glutathione adjusted to pH 7.6 (final volume, 2 ml), pH 7.6, 37O, 0,. At the end of 1 hr the reaction was stopped by the addition of 1 ml of 10% TCA and the activity was measured by ascorbate production. The activity has been defined as @moles of ascorbic acid formed per milligram of protein per hour under assay conditions. Dehydroascorbatase activity was estimated by the method of Kagawa et al.10 The incubation mixture consisted of soluble fraction equivalent to 100 mg of tissue, 10 pmoles of dehydroascorbate (freshly prepared by bromine oxidation of ascorbate), 0.3 pmoles of glutathione in Tris-malate buffer (200 pmoles), pH 6.8, (final volume, 3 ml), 37”. The reaction was stopped after 5 min of incubation by 1 ml of 20% metaphosphoric acid and 2% SnCl,, and the remaining dehydroascorbic acid was rapidly reduced with H,S. A O.lml sample was taken for the estimation of 2,3 diketogulonic acid formed in the system. The specific activity has been defined as pmoles of diketogulonate formed per milligram of protein per 5 min under assay conditions. 2,3 Diketoaldonate decarboxylase activity was estimated by the method of Kagawa.5 The incubation mixture consisted of soluble fraction equivalent to 100 mg of the tissue, 10 @moles of diketogulonate, 25 pmoles of phosphate buffer pH 6.8 (final, volume 2.5 ml), 37O, N?. The enzyme activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as @liters of CO, evolved per milligram of protein at 30-min intervals under assay conditions. Protein was determined by the method of Lowry et al.,11 using bovine serum albumin as the standard. RESULTS As shown in Table 1, there were slight increase in the contents of ascorbic acid (p < 0.02) and dehydroascorbic acid (p < 0.05) of liver 1 hr following
to normal in the
NATHAN1 AND NATH
Table 2.-Effect of Corticotropin on Activities of Ascorbic Acid Synthesizing Enzymes in Liver of Normal and Adrenalectomized Rats ._. __ Soecific Groups
Normal Rats 1 2 Saline 3 Corticotropin Adrenalectomized Rats 4 Saline 5 Corticotropin
Values are expressed in text.
940 * 27 955 f 30 973 f 45
1341 * 43 1350 f 37 1367 ?a 48
220 + 13 208 r 19 233 + 19
1.26 1.23 1.27
r 0.019 -t 0.017 -c 0.010
7.56 it: 0.05 7.51 t 0.08 7.59 + 0.06
710 * 35 721 rt 48
1381 + 60 1370 2 51
198 + 21 209 + 18
0.947 ? 0.012 0.940 * 0.008
6.25 + 0.09 6.13 r 0.09
as mean k SEM of six rats. Respective
enzyme units are denoted
caused a moderate but significant increase (p < 0.001) in the total ascorbic acid content of blood. It is evident from Table 1 that corticotropin had no appreciable effect on the contents of liver ascorbic acid, dehydroascorbic acid, and diketogulonic acid or on total ascorbic acid content of blood in adrenalectomized rats as compared to its control (Group 4). It is evident from Table 2 that corticotropin had no significant effect on any of the ascorbic acid synthesizing enzymes (D-glucurono-&lactone hydrolase, L-gulono-y-lactone hydrolase, TPN-L-hexonate dehydrogenase, and L-gulono-y-lactone oxidase) in intact and adrenalectomized rats as compared to their controls. It is interesting to note that there was a marked decrease (p < 0.02) in the activity of dehydroascorbatase in liver and kidney. Under similar conditions, the decrease in the activity of another degrading enzyme, 2,3 diketoaldonate decarboxylase, was moderate but significant (p < 0.001) in liver and kidney. These enzymes were not affected by corticotropin in adrenalectomized rats (Table 3). DISCUSSION It is evident from the results that corticotropin administration caused a significant alteration in the ascorbic acid metabolism in rats. The changes in the
Table 3 .-Effect of Corticotropin on Activities of Ascorbic Acid Degrading Enzymes in Liver and Kidney of Normal and Adrenalectomized Rats Specific
Normal Rats 1 2 Saline 3 Corticotropin Adrenalectomized Rats 4 Saline 5 Corticotropin
2,3 Diketoaldonate Decarboxvlase
2,3 Diketoaldonate Decarboxvlase
0.107 + 0.013 0.109 + 0.011 0.043 f 0.012*
12.63 -F 0.63 12.79 c 0.71 8.47 r 0.94 t
0.048 + 0.006 0.052 f 0.007 0.031 r 0.003*
5.8 t 0.07 5.92 ? 0.14 4.10 r 0.17
0.257 -c 0.015 0.264 f 0.019
14.68 + 0.52 14.35 f 0.61
0.087 z? 0.006 0.091 * 0.010
6.50 -c 0.19 6.17 e 0.30
Respective enzyme units are denoted in text. Values are expressed six rats. * p < 0.02 as compared to Group 1 using Student’s t test. t p < 0.001 as compared to Group 1 using Student’s t test.
as mean 2 SEM of
CORTICOTROPIN AND ASCORBIC ACID
ascorbic acid content in corticotropin-administered rats reported in the present communication are in accordance with the results of earlier investigators.3J2 Our results show that corticotropin has no effect on ascorbic acid biosynthesis in intact rats, as it has no appreciable effect on any of the ascorbic acid synthesizing enzymes in liver. However, the catabolism of ascorbic acid was found to be affected considerably by corticotropin. There was a marked decrease in the activities of ascorbic acid catabolizing enzymes such as dehydroascorbatase and 2,3 diketoaldonate decarboxylase, both in liver and kidney. This also accounts for the slight increase in dehydroascorbic acid and decrease in diketoguionic acid content of liver in corticotropin-administered rats. There is a possibility of the conversion of glucose to glucuronate through the influence of corticotropin and this might be a step-limiting factor in the formation of ascorbic acid. This may also account partially for the increase in ascorbic acid by corticotropin, but this possibility needs further investigation. It is also clear from our results that corticotropin works indirectly through the adrenocortical axis, as it has no effect on ascorbic acid metabolism in adrenalectomized rats. Lahiri and Lloyd,12 who studied the effect of corticotropin on the contents of ascorbic acid and dehydroascrobic acid in rats, are of the opinion that corticotropin alters ascorbic acid metabolism by decreasing ascorbic acid catabolism. Salomon and Stubbsl” have demonstrated that corticotropin depresses ascorbic acid catabolism as judged by the decrease in respiratory radioactivity originating from ascorbic-l-14C acid. Recently it has been reported from this laboratory that adrenalectomy affects ascorbic acid metabolism mainly by increasing the activity of ascorbic acid degrading enzymes. In view of these findings it can be suggested that corticotropin affects ascorbic acid metabolism by decreasing the activities of dehydroascorbatase and 2,3 diketoaldonate decarboxylase in liver and kidney. ACKNOWLEDGMENT The authors suggestions.
to Dr. N. Nath and Dr. H. F. Daginawala
for their valuable
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6. Oser, B. L. (Ed.): Hawk’s Physiological Chemistry (ed. 14). New York, McGraw-Hill, 1965. 7. Row, J. H., Mills, M. B., Osterling, M. J., and Damron, C. M.: The determination of Diketo-Ggulonic acid, dehydro-Lascorbic acid and L-ascorbic acid in the same tissue extract by the 2,4 dinitrophenylhydrazine method. J. Biol. Chem. 174:201, 1948. 8. Salomon, L. L., and Stubbs, D. W.: The defect in ascorbate synthesis by hypopphysectomized rats. Biochem. Biophys. Res. Comm. 4:239, 1961.
142 9. -, and -: Restoration of aldonolactonase activity by somatotropin in hypophysectomized rats. Biochem. Biophys. Res. Comm. 5:349, 1961. 10. Kagawa, Y., Takiguchi, H., and Shimagano, N. : Enzymatic delactonization of dehydro-L-ascorbate in animal tissues. Biochem. Biophys. Acta 51:413, 1961. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. E., and Randall, R. J.: Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. 12. Lahiri, S., and Lloyed, B. B.: The :ffect of stress and corticotropin on the concentrations of vitamin C in blood and tissue of the Rat. Biochem. J. 84:478, 1962. 13. Salomon, L. L., and Stubbs, D. W.: Some aspects of the metabolism of ascorbic acid in rats. Ann. N. Y. Acad. Sci. 92:128, 1961.