- Email: [email protected]
Ascorbic acid metabolism in adrenalectomized rats
Ascorbic Acid Metabolism in Adrenalectomized Rats By MAHESHG. NATHANI,IDREESBHAI, N. NATH, H. F. DAGINAWALA, ANY
M. C. NATH Male albino rats were adrenalectomized and their ascorbic acid, dehydroascorbic acid, and diketogulonic acid were determined in liver and urine, while only the total ascorbic acid was determined in blood, at the intervals of 4, 8, and 12 days after adrenalectomy. Activities of ascorbic acid synthesizing enzyme Dglucurono-6-lactone hydrolase, L-gulonoy-lactone hydrolase, and L-gulono-y-lactone oxidase were estimated in liver. The activities of ascorbic acid degrading enzymes dehydroascorbatase and 2,3 diketoaldonate decarbozylase were studied in liver and kidney. In adrenalectomized rats there was a slight decrease in the ascorbic acid level and an appreciable decrease in dehydroascorbic acid content of the liver. On the other hand, there was about twofold increase in the diketogtdonic acid
content of liver. The content of ascorbic acid and dehydroascorbic acid in the urine of adrenalectomized rats decreased significantly, while there was slight increase in the diketogulonic acid content. There was also a slight decrease in total ascorbic acid content of blood. Decreases of about 20% and 30% were observed in the activities of u-glucurono-Nactone hydrolase and L-gulono-y-lactone oxidase respectively, while the activity of Lhydrolase remained gulono-y-lactone practically unchanged. An appreciable increase in the activity of dehydroascorbatase and a moderate increase in the activity of diketoaldonate decarboxylase were observed in liver and kidney of adrenalectomized rats. All corresponding alterations were more conspicuous in rats 12 days after adrenalectomy.
I
T HAS BEEN ESTABLISHED by earlier workers that certain hormones play important roles in the metabolism of ascorbic acid. Synthesis of ascorbic acid in liver is known to be diminished in hypophysectomized rats,ls3 and administration of growth hormone to these rats has been known to increase the synthesis of ascorbic acid.” It has also been reported that the stimulation of the pituitary-adrenal axis increases the blood ascorbic acid.4 Corticotropin has been reported to increase the level of ascorbic acid in liver and blood.5.0 Various studies have indicated that ascorbic acid metabolism is closely associated with the metabolism of the adrenal steroids. Ascorbic acid has been From India.
the
Received Supported
University
Department
for publication
May
by the India Council
of Biochemistry,
21,
Mahatma
Gandhi
Marg,
Nagpur,
1971.
of Medical
Research,
India.
MAHESH G. NATHANI, M.Sc.: Research Fellow, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. IDREES BHAI, M.Sc.: Lecturer in Biochemistry, Corporation Medical College, Nagpur, India. N. NATH, MSc., PH.D.: Lecturer in Biochemistry, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. University Department of BioH. F. DAGINAWALA, PH.D.: Lecturer in Biochemistry, chemistry, Mahatma Gandhi Marg, Nagpur, India. M. C. NATH, D.Sc., F.N.I., F.R.I.C.: Professor and Head, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. 1036
METABOLISM. VOL. 20, No.
11 (NOVEMBER), 1971
ASCORBIC
1037
ACID METABOLISM
shown to accelerate certain reactions of steroid synthesis in vitro and to slow down some others.7 This vitamin is also known to inhibit the action of several stress agents.7 Although ascorbic acid is present in relatively large amounts in the adrenals, its definite role in the function of adrenals has not yet been established. The action of different adrenal hormones on the activities of synthesizing and degrading enzymes of ascorbic acid has also not been studied. It was felt necessary, therefore, to examine the effect of adrenalectomy on ascorbic acid, dehydroascorbic acid, and diketogulonic acid contents of liver, blood, and urine, and also on the metabolism of ascorbic acid in rat liver and kidney. MATERIALS
AND
METHODS
n-Glucuronic acid lactone was obtained from Sigma Chemical Co. Dr. Stubbs (Department of Physiology, University of Texas Medical Center, Galveston, Texas) kindly supplied the L-gulono-y-lactone. 2,3 Diketogulonate was prepared by the method of Kagawa.8 Male albino rats (100-125 g body wt) were maintained on stock laboratory dieta and distributed into two groups. Bilateral adrenalectomy was carried out for group II rats, while rats of group I were sham-operated controls. The experiments were carried out after 4, 8, and 12 days of adrenalectomy. Rats were killed by decapitation, and blood was collected in oxalated tubes for total ascorbic acid estimation. Liver and kidneys were removed, rinsed in ice-cold water, and blotted dry. Part of the tissue was homogenized in 9 volumes of isotonic sucrose. The homogenate was centrifuged at 10,000 g for 20 min to obtain tissue extract free of heavy particles as the supernate. The supernate thus obtained was centrifuged at 100,000 g for 1 hr to yield microsomes and soluble fraction. The other part of the tissue was homogenized in 4 volumes of isotonic sucrose, and the homogenate obtained was used for the assay of complete system. The following different estimations were carried out.
Ascorbic Acid, Dehydroascorbic Acid, and Diketogulonic Acid These were estimated in liver, blood, and urine oxalic acid) by the method of Roe et al.10
D-Glucurono-&Lactone
(24-hr urine samples
collected
in 10%
Hydrolase (EC 3.1 .1.19)
The activity was estimated by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: microsomes equivalent to 8.75 mg liver, 0.04 M NaHCOs, 0.04 M n-glucurono-S-lactone, (final volume 2 ml), pH 7.6, 25V, 95% N,-5% CO,. Activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as the evolution of Jiters of CO, per mg protein at 30min intervals under the assay conditions.
L-Gulono-y-Lactone
Hydrolase (EC 3.1 .1.18)
The activity was estimated by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: soluble fraction equivalent to 3.75 mg liver, 0.04 M NaHCO,, 0.07 M L-gulano-y-lactone, (final volume 1 ml), pH 7, 6, 25’C, 95% N,-5% CO.,. Activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as the evolution of pliters of CO, per mg protein at 30-min intervals under the assay conditions.
L-Gulono-y-Lactone
Oxidase (L-Gulono-y-Lactone
Oxidoreductase EC I .I .3.8)
The activity was estimated by the modified method of Salomon and Stubbs.11 The incubation mixture consisted of the following: microsomes suspended in 0.05 M Tris (hydroxy-methyl) aminomethane-HCl buffer, pH 7,6. equivalent to 120 mg liver; 0.00042
Values are * p < 0.001 i p < 0.01 $ p < 0.02
Liver
-c f f i-
0.06 0.03 0.07 0.073
1.620 + 2.360* f 3.310*+ 3.490*-r-
six rats. using Student’s t test. using Student’s t test. using Student’s t test.
4.75 2.36* 1.39* 1.12* 0.012 0.017 0.016 0.017
Dehydroascorbic Acid DiketogulonicAcid (mg/lOO g fresh liver wt)
mean +- SEM of to sham controls to sham controls to sham controls
20.105 f. 0.5 18.538 z!I 0.38 17.735-F * 0.590 17.320t k 0.710
-Ascorbic Acid
expressed as as compared as compared as compared
Sham controls 4 8 12
Days After Adrenalectomy
23.675 c 18.314$k 11.600tzk 10.51Oi +
1.120 1.5 2.250 2.700
Ascorbic Acid
10.32 + 7.75 + 4.28$ + 2.17t+
1.30 0.98 1.50 1.70
Dehydroascorbic Acid (mg/lOO ml urine)
Urine
5.17 6.32* 6.37* 6.89*
f + + +
0.09 0.07 0.10 0.06
Diketogulonic Acid
Table l.-Ascorbic Acid, Dehydroascorbic Acid, and Diketogulonic Acid Contents of Liver, Urine, and Blood in Adrenalectomized Rats Blood
1.250 0.995* 0.956* 0.952*
+ + + +
0.017 0.011 0.014 0.010
Total Ascorbic Acid (mg/lOO ml blood)
1039
ASCORBIC ACID METABOLISM
M r_-gulono-y-lactone, 0.02 M glutathione adjusted to pH 7.6 with NaHCO, (all concentrations final), total volume 2.0 ml, were incubated for 1 hr under 0, at 25’C. After 1 hr, 1 ml of 10% trichloroacetic acid (TCA) was added to stop the reaction, and the activity was measured by ascorbate production .lO The specific activity has been defined as &moles of ascorbic acid formed/mg protein per hr under the assay conditions.
Assay of Complete System This is assayed by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: liver extract equivalent to 120 mg liver, 0.05 M Tris (hydroxymethyl) aminomethane-HCI buffer, 0.002 M sodium-mglucuronate 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, 37’C!, 0,. Reaction was stopped by addition of 1 ml 10% TCA after 1 hr, and the activity was measured by ascorbate production. The activity has been defined as pmoles of ascorbic acid formed/mg protein per hr under the assay conditions.
Dehydroascorbatase The activity was estimated by the method of Kagawa et al.12 The incubation mixture consisted of the following: soluble fraction equivalent to 100 mg of tissue, 10 pmoles of dehydroascorbate (freshly prepared by bromine oxidation of ascorbate), 0.3 &moles of glutathione in Tris-malate buffer (200 amoles) pH 6.8, (final volume 3.0 ml), 37OC. The reaction was stopped after 5 min of incubation by 1.0 ml of 20% metaphosphoric acid + 2% SnCl,, and the remaining dehydroascorbic acid was rapidly reduced with H,S. The specific activity has been defined as pmoles of diketogulonate formed/mg of protein per 5 min under the assay conditions.
2,3 Diketoaldonate
Decarboxylase
The activity was estimated by the method of Kagawa.8 The incubation mixture consisted of the following: soluble fraction equivalent to 100 mg of the tissue, 0.00025 M dikeogulonate, 0.025 M phosphate buffer pH 6.8, (final volume 2.5 ml), 37OC, N,. The enzyme activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as &liters of CO, evolved/mg protein at 30-min intervals under the assay conditions.
Protein It was determined standard.
by the method
of Lowry et al.13 using bovine serum albumin
as the
RESULTS
As shown in Table 1, there was a slight decrease in the ascorbic acid (reduced) content of liver following adrenalectomy. A decrease of about 8% was Table Z.-Effect
Days After Adrenalectomy
Sham Controls 4 8 12
of Adrenalectomy on Activities of Some Ascorbic Acid Synthesizing Enzymes in Rat Liver _. ^_ P-tilUCUrOno-o-LaCtone L-GUlOIlO-y-La&me L-Gulono-y-La&one Hydrolase.
Hydrolase
Oxidase
Specific Activity
960 776 701 686
-c + f ?
20 16* 27* 18*
1355 1320 1397 1356
+ 34 rt 37 +- 29 f 21
1.353 1.070 0.922 0.903
+- 0.008 & 0.010” c 0.014* f 0.120*
Values are expressed as mean 2 SEM of six rats. Respective enzyme activities are denoted in text. * p < 0.001 as compared to sham controls using Student’s t test.
Total System
7.94 6.28 6.21 6.10
2 f r -c
0.05 0.07* 0.067* 0.063*
1040
NATHAN1
ET
AL.
Fig. l.-Effect of adrenalectomy on activity of hepatic n-glucurono-d-lactone hydrolase. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
after 4 days of adrenalectomy, which went down to about 15% after 12 days of adrenalectomy. However, the decrease observed after 8 days of adrenalectomy was quite significant as shown by the p values (Table 1) . On the other hand, there was decrease in dehydroascorbic acid content of liver even after 4 days of adrenalectomy. Simultaneously, there was increase in the diketogulo’nic acid content of liver, under similar conditions. Ascorbic acid and dehydroascorbic acid contents of urine of adrenalectomized rats decreased by about 50% and 75% respectively, following 12 days of adrenalectomy. Diketogulonic acid content of urine was significantly increased, although the percentage increase was moderate. A moderate but significant decrease was observed in the total ascorbic acid content (ascorbic acid + dehydroascorbic acid + diketogulonic acid) of blood. The results are shown in Table 1. Results showing the effect of adrenalectomy on hepatic ascorbic acid synthesizing enzymes are presented in Table 2. Adrenalectomy caused a decrease in the activity of D-glucurono-S-lactone hydrolase (Fig. 1) and L-gulono-y-lactone oxidase by about 20% and 30%, respectively. However, under similar conditions, the activity of L-gulono-y-lactone hydrolase remained practically unchanged (Fig. 2).
observed
Fig. Z.-Effect of adrenalectomy on activity of hepatic L-gulono-y-lactone hydrolase. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
1041
ASCORBIC ACID METABOLISM
Table 3.--Et&t of Adrenalectomy on Activities of Ascorbic Acid Degrading Enzymes in Liver and Kidney Kidney
Liver Dehydroascorbatase
Days After Adrenalectomy
Sham Controls 4 8 12
2,3 Diketoaldonate Decarboxylase Soecific
Dehydroascorbatase
0.045 5 0.001 0.70 0.060 r O.OOl* 0.41$ 0.093 It 0.003* 0.57t 0.0904 + 0.002* 0.68t _____ Numerals in parentheses represent number of rats used. Respective enzyme activities are denoted in text. * p < 0.001 as compared to sham controls using Student’s t test. i p < 0.01 as compared to sham controls using Student’s t test. Z p < 0.02 as compared to sham controls using Student’s t test. (6) (4) (6) (5)
0.106 0.187 0.252 0.261
f 5 k +
0.008 0.006’ 0.012* 0.014*
12.50 13.28 14.82 15.66
+ 1 f -c
2,3 Diketoaldonate Decarboxylase
Activity
5.4 5.8 6.4 6.85
+ 0.080 -c 0.063* r 0.090* +- 0.076”
A marked increase of about 60% was observed in the activity of ascorbic acid degrading enzyme, dehydroascorbatase, both in liver and kidney after 12 days of adrenalectomy (Table 3). The increase in the activity of 2,3 diketoaldonate decarboxylase was moderate but highly significant in kidney (Figs. 3 and 4). DISCUSSION
It is apparent from the results that adrenalectomy caused some alteration in the ascorbic acid metabolism in rats. The decrease in the ascorbic acid content of liver in adrenalectomized rats, reported in the present communication, is in accordance with the results of earlier investigators.14J5 Results reported by Shamrai et al.16 are slightly different from those of ours. Their results show that ascorbic acid remained practically unchanged, while dehydroascorbic acid disappeared almost completely and diketogulonic acid increased two to three times in adrenalectomized rats. Dumm and RaliP have reported that adrenalectomy decreases the urinary excretion of ascorbic acid in rats, which is in accordance with our results (Table 1) . Our results also show that adrenalectomy is having certain specific effects on both the biosynthesis and the catabolism of ascorbic acid in rats. A moderate decrease in the formation of ascorbic acid by the total system shows that adrenalectomy diminishes the synthesis of this vitamin in the rat liver. This
Fig. 3.-Effect of adrenalectomy on activity of 2.4 diketoaldonate decarbozylase in liver. A-enzyme activity of shamoperated controls; B, C, D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
f 20e
1042
NATHAN1 ET AL.
Fig. 4.-Effect of adrenalectomy on activity of 2,4 diketoaldonate decarboxylase in kidney. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
change can be explained on the basis of the decrease found in the activities of ascorbic acid synthesizing enzymes such as b-glucurono-Slactone hydrolase and L-gulono-y-lactone oxidase in rat liver. However, it seems that the catabolism of ascorbic acid is affected most by adrenalectomy (Table 3). There is a marked increase in the activity of dehydroascorbatase both in the liver and the kidney. The other enzyme, 2,3 diketoaldonate decarboxylase, also shows moderate increase. This explains partially, the depletion of dehydroascorbic acid and accumulation of diketogulonic acid in liver of adrenalectomized rats. REFERENCES 1. 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. 2. - and -: The defect in ascorbic synthesis by hypophysectomized rats, Biochem. Biophys. Res. Commun. 4:239, 1961. 3. -, -, and Daginawals, H. F.: Effects of hormones and drugs on activities of Lgulono-y-lactone hydrolase. Proc. Sot. Exp. Biol. Med. 115:800, 1954. 4. Allison, J. E.: Total plasma ascorbic acid as index of stress in rat. Proc. Sot. Exp. Biol. Med. 90:277, 1955. 5. Stewart, C. P., Horn, D. B., and Robson, J. S.: Effect of cortisone and adrenocorticotropic hormones on the dehydroascorbic acid of human plasma. Biochem. J. 53:254, 1953. 6. Sinha, C., and Lahiri, S.: Effect of corticotropin on the concentration of vitamin C in tissues of mice. Indian J. Exp. Biol. 2: 102, 1964. 7. Chelopin, H., Mouton, M., and Ratsimarnanga, A. R.: Some interrelation between ascorbic acid and adrenocortical function. World Rev. Nutr. Diet. 6: 165, 1966. 8. Kagawa, Y.: Enzymatic studies on ascorbic acid catabolism in animals. J. Biothem. (Tokyo) 51:134, 1962.
9. User, B. L.: Hawk’s Physiological Chemistry (ed. 14). New York, McGraw Hill, 1965, p. 1121. 10. Roe, J. H., Mills, M. B., Osterling, M. J., and Damron, C. M.: The determination of Diketo-r_-gulonic acid, dehydro-r. ascorbic acid and L-ascorbic acid in the same tissue extract by the 2,4 dinitrophyenylhydrazine method. J. Biol. Chem. 174:201, 1948. 11. Salomon, L. L., and Stubbs, D. W.: Restoration of aldonolactonase activity by somatotropin in hypophysectomized rats. Biochem. Biophys. Res. Commun. 5:349, 1961. 12. Kagawa, Y., Takiguchi, H., and Shimagono, N.: Enzymatic delactonization of Dehydro-L-ascorbate in animal tissues. Biochim. Biophys. Acta 51:413, 1961. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. 14. Giovanni, C., Gerruccio, Di. So, and Andrea, L.: The existence in the rat liver of a probable combined form of vitamin C and its behavior after adrenalectomy. Atti. Sot. Peloritana Sci. Fis. Mat. Natur. 4:289, 1957.
1043
ASCORBIC ACID METABOLISM
15. Cuzzocrea, G., Stefano, F. De., and Lino, A.: Free ascorbic acid in the liver of adrenalectomized rats. Sperimentale 109 : 4 13, 1959. 16. Shamrai, Plattonov, of ascorbic
E. F., Ivanyuta,
0. M.: Contents
0. M., and
of various forms
acid in the tissues of adrenalec-
tomized animals. Ukr. Biokhim. Zh. 37:268, 1965. 17. Dumm, M. E., and Ralli, E. P.: The excretion of pantothenic acid and ascorbic acid by intact and adrenalectomized rats on diets supplemented with and deficient in, pantothenic acid. Endocrinology 45: 188, 1949.
M. C. NATH Male albino rats were adrenalectomized and their ascorbic acid, dehydroascorbic acid, and diketogulonic acid were determined in liver and urine, while only the total ascorbic acid was determined in blood, at the intervals of 4, 8, and 12 days after adrenalectomy. Activities of ascorbic acid synthesizing enzyme Dglucurono-6-lactone hydrolase, L-gulonoy-lactone hydrolase, and L-gulono-y-lactone oxidase were estimated in liver. The activities of ascorbic acid degrading enzymes dehydroascorbatase and 2,3 diketoaldonate decarbozylase were studied in liver and kidney. In adrenalectomized rats there was a slight decrease in the ascorbic acid level and an appreciable decrease in dehydroascorbic acid content of the liver. On the other hand, there was about twofold increase in the diketogtdonic acid
content of liver. The content of ascorbic acid and dehydroascorbic acid in the urine of adrenalectomized rats decreased significantly, while there was slight increase in the diketogulonic acid content. There was also a slight decrease in total ascorbic acid content of blood. Decreases of about 20% and 30% were observed in the activities of u-glucurono-Nactone hydrolase and L-gulono-y-lactone oxidase respectively, while the activity of Lhydrolase remained gulono-y-lactone practically unchanged. An appreciable increase in the activity of dehydroascorbatase and a moderate increase in the activity of diketoaldonate decarboxylase were observed in liver and kidney of adrenalectomized rats. All corresponding alterations were more conspicuous in rats 12 days after adrenalectomy.
I
T HAS BEEN ESTABLISHED by earlier workers that certain hormones play important roles in the metabolism of ascorbic acid. Synthesis of ascorbic acid in liver is known to be diminished in hypophysectomized rats,ls3 and administration of growth hormone to these rats has been known to increase the synthesis of ascorbic acid.” It has also been reported that the stimulation of the pituitary-adrenal axis increases the blood ascorbic acid.4 Corticotropin has been reported to increase the level of ascorbic acid in liver and blood.5.0 Various studies have indicated that ascorbic acid metabolism is closely associated with the metabolism of the adrenal steroids. Ascorbic acid has been From India.
the
Received Supported
University
Department
for publication
May
by the India Council
of Biochemistry,
21,
Mahatma
Gandhi
Marg,
Nagpur,
1971.
of Medical
Research,
India.
MAHESH G. NATHANI, M.Sc.: Research Fellow, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. IDREES BHAI, M.Sc.: Lecturer in Biochemistry, Corporation Medical College, Nagpur, India. N. NATH, MSc., PH.D.: Lecturer in Biochemistry, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. University Department of BioH. F. DAGINAWALA, PH.D.: Lecturer in Biochemistry, chemistry, Mahatma Gandhi Marg, Nagpur, India. M. C. NATH, D.Sc., F.N.I., F.R.I.C.: Professor and Head, University Department of Biochemistry, Mahatma Gandhi Marg, Nagpur, India. 1036
METABOLISM. VOL. 20, No.
11 (NOVEMBER), 1971
ASCORBIC
1037
ACID METABOLISM
shown to accelerate certain reactions of steroid synthesis in vitro and to slow down some others.7 This vitamin is also known to inhibit the action of several stress agents.7 Although ascorbic acid is present in relatively large amounts in the adrenals, its definite role in the function of adrenals has not yet been established. The action of different adrenal hormones on the activities of synthesizing and degrading enzymes of ascorbic acid has also not been studied. It was felt necessary, therefore, to examine the effect of adrenalectomy on ascorbic acid, dehydroascorbic acid, and diketogulonic acid contents of liver, blood, and urine, and also on the metabolism of ascorbic acid in rat liver and kidney. MATERIALS
AND
METHODS
n-Glucuronic acid lactone was obtained from Sigma Chemical Co. Dr. Stubbs (Department of Physiology, University of Texas Medical Center, Galveston, Texas) kindly supplied the L-gulono-y-lactone. 2,3 Diketogulonate was prepared by the method of Kagawa.8 Male albino rats (100-125 g body wt) were maintained on stock laboratory dieta and distributed into two groups. Bilateral adrenalectomy was carried out for group II rats, while rats of group I were sham-operated controls. The experiments were carried out after 4, 8, and 12 days of adrenalectomy. Rats were killed by decapitation, and blood was collected in oxalated tubes for total ascorbic acid estimation. Liver and kidneys were removed, rinsed in ice-cold water, and blotted dry. Part of the tissue was homogenized in 9 volumes of isotonic sucrose. The homogenate was centrifuged at 10,000 g for 20 min to obtain tissue extract free of heavy particles as the supernate. The supernate thus obtained was centrifuged at 100,000 g for 1 hr to yield microsomes and soluble fraction. The other part of the tissue was homogenized in 4 volumes of isotonic sucrose, and the homogenate obtained was used for the assay of complete system. The following different estimations were carried out.
Ascorbic Acid, Dehydroascorbic Acid, and Diketogulonic Acid These were estimated in liver, blood, and urine oxalic acid) by the method of Roe et al.10
D-Glucurono-&Lactone
(24-hr urine samples
collected
in 10%
Hydrolase (EC 3.1 .1.19)
The activity was estimated by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: microsomes equivalent to 8.75 mg liver, 0.04 M NaHCOs, 0.04 M n-glucurono-S-lactone, (final volume 2 ml), pH 7.6, 25V, 95% N,-5% CO,. Activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as the evolution of Jiters of CO, per mg protein at 30min intervals under the assay conditions.
L-Gulono-y-Lactone
Hydrolase (EC 3.1 .1.18)
The activity was estimated by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: soluble fraction equivalent to 3.75 mg liver, 0.04 M NaHCO,, 0.07 M L-gulano-y-lactone, (final volume 1 ml), pH 7, 6, 25’C, 95% N,-5% CO.,. Activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as the evolution of pliters of CO, per mg protein at 30-min intervals under the assay conditions.
L-Gulono-y-Lactone
Oxidase (L-Gulono-y-Lactone
Oxidoreductase EC I .I .3.8)
The activity was estimated by the modified method of Salomon and Stubbs.11 The incubation mixture consisted of the following: microsomes suspended in 0.05 M Tris (hydroxy-methyl) aminomethane-HCl buffer, pH 7,6. equivalent to 120 mg liver; 0.00042
Values are * p < 0.001 i p < 0.01 $ p < 0.02
Liver
-c f f i-
0.06 0.03 0.07 0.073
1.620 + 2.360* f 3.310*+ 3.490*-r-
six rats. using Student’s t test. using Student’s t test. using Student’s t test.
4.75 2.36* 1.39* 1.12* 0.012 0.017 0.016 0.017
Dehydroascorbic Acid DiketogulonicAcid (mg/lOO g fresh liver wt)
mean +- SEM of to sham controls to sham controls to sham controls
20.105 f. 0.5 18.538 z!I 0.38 17.735-F * 0.590 17.320t k 0.710
-Ascorbic Acid
expressed as as compared as compared as compared
Sham controls 4 8 12
Days After Adrenalectomy
23.675 c 18.314$k 11.600tzk 10.51Oi +
1.120 1.5 2.250 2.700
Ascorbic Acid
10.32 + 7.75 + 4.28$ + 2.17t+
1.30 0.98 1.50 1.70
Dehydroascorbic Acid (mg/lOO ml urine)
Urine
5.17 6.32* 6.37* 6.89*
f + + +
0.09 0.07 0.10 0.06
Diketogulonic Acid
Table l.-Ascorbic Acid, Dehydroascorbic Acid, and Diketogulonic Acid Contents of Liver, Urine, and Blood in Adrenalectomized Rats Blood
1.250 0.995* 0.956* 0.952*
+ + + +
0.017 0.011 0.014 0.010
Total Ascorbic Acid (mg/lOO ml blood)
1039
ASCORBIC ACID METABOLISM
M r_-gulono-y-lactone, 0.02 M glutathione adjusted to pH 7.6 with NaHCO, (all concentrations final), total volume 2.0 ml, were incubated for 1 hr under 0, at 25’C. After 1 hr, 1 ml of 10% trichloroacetic acid (TCA) was added to stop the reaction, and the activity was measured by ascorbate production .lO The specific activity has been defined as &moles of ascorbic acid formed/mg protein per hr under the assay conditions.
Assay of Complete System This is assayed by the modified method of Salomon and Stubbs.2 The incubation mixture consisted of the following: liver extract equivalent to 120 mg liver, 0.05 M Tris (hydroxymethyl) aminomethane-HCI buffer, 0.002 M sodium-mglucuronate 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, 37’C!, 0,. Reaction was stopped by addition of 1 ml 10% TCA after 1 hr, and the activity was measured by ascorbate production. The activity has been defined as pmoles of ascorbic acid formed/mg protein per hr under the assay conditions.
Dehydroascorbatase The activity was estimated by the method of Kagawa et al.12 The incubation mixture consisted of the following: soluble fraction equivalent to 100 mg of tissue, 10 pmoles of dehydroascorbate (freshly prepared by bromine oxidation of ascorbate), 0.3 &moles of glutathione in Tris-malate buffer (200 amoles) pH 6.8, (final volume 3.0 ml), 37OC. The reaction was stopped after 5 min of incubation by 1.0 ml of 20% metaphosphoric acid + 2% SnCl,, and the remaining dehydroascorbic acid was rapidly reduced with H,S. The specific activity has been defined as pmoles of diketogulonate formed/mg of protein per 5 min under the assay conditions.
2,3 Diketoaldonate
Decarboxylase
The activity was estimated by the method of Kagawa.8 The incubation mixture consisted of the following: soluble fraction equivalent to 100 mg of the tissue, 0.00025 M dikeogulonate, 0.025 M phosphate buffer pH 6.8, (final volume 2.5 ml), 37OC, N,. The enzyme activity was determined manometrically by CO, evolution for 1 hr. The specific activity has been defined as &liters of CO, evolved/mg protein at 30-min intervals under the assay conditions.
Protein It was determined standard.
by the method
of Lowry et al.13 using bovine serum albumin
as the
RESULTS
As shown in Table 1, there was a slight decrease in the ascorbic acid (reduced) content of liver following adrenalectomy. A decrease of about 8% was Table Z.-Effect
Days After Adrenalectomy
Sham Controls 4 8 12
of Adrenalectomy on Activities of Some Ascorbic Acid Synthesizing Enzymes in Rat Liver _. ^_ P-tilUCUrOno-o-LaCtone L-GUlOIlO-y-La&me L-Gulono-y-La&one Hydrolase.
Hydrolase
Oxidase
Specific Activity
960 776 701 686
-c + f ?
20 16* 27* 18*
1355 1320 1397 1356
+ 34 rt 37 +- 29 f 21
1.353 1.070 0.922 0.903
+- 0.008 & 0.010” c 0.014* f 0.120*
Values are expressed as mean 2 SEM of six rats. Respective enzyme activities are denoted in text. * p < 0.001 as compared to sham controls using Student’s t test.
Total System
7.94 6.28 6.21 6.10
2 f r -c
0.05 0.07* 0.067* 0.063*
1040
NATHAN1
ET
AL.
Fig. l.-Effect of adrenalectomy on activity of hepatic n-glucurono-d-lactone hydrolase. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
after 4 days of adrenalectomy, which went down to about 15% after 12 days of adrenalectomy. However, the decrease observed after 8 days of adrenalectomy was quite significant as shown by the p values (Table 1) . On the other hand, there was decrease in dehydroascorbic acid content of liver even after 4 days of adrenalectomy. Simultaneously, there was increase in the diketogulo’nic acid content of liver, under similar conditions. Ascorbic acid and dehydroascorbic acid contents of urine of adrenalectomized rats decreased by about 50% and 75% respectively, following 12 days of adrenalectomy. Diketogulonic acid content of urine was significantly increased, although the percentage increase was moderate. A moderate but significant decrease was observed in the total ascorbic acid content (ascorbic acid + dehydroascorbic acid + diketogulonic acid) of blood. The results are shown in Table 1. Results showing the effect of adrenalectomy on hepatic ascorbic acid synthesizing enzymes are presented in Table 2. Adrenalectomy caused a decrease in the activity of D-glucurono-S-lactone hydrolase (Fig. 1) and L-gulono-y-lactone oxidase by about 20% and 30%, respectively. However, under similar conditions, the activity of L-gulono-y-lactone hydrolase remained practically unchanged (Fig. 2).
observed
Fig. Z.-Effect of adrenalectomy on activity of hepatic L-gulono-y-lactone hydrolase. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
1041
ASCORBIC ACID METABOLISM
Table 3.--Et&t of Adrenalectomy on Activities of Ascorbic Acid Degrading Enzymes in Liver and Kidney Kidney
Liver Dehydroascorbatase
Days After Adrenalectomy
Sham Controls 4 8 12
2,3 Diketoaldonate Decarboxylase Soecific
Dehydroascorbatase
0.045 5 0.001 0.70 0.060 r O.OOl* 0.41$ 0.093 It 0.003* 0.57t 0.0904 + 0.002* 0.68t _____ Numerals in parentheses represent number of rats used. Respective enzyme activities are denoted in text. * p < 0.001 as compared to sham controls using Student’s t test. i p < 0.01 as compared to sham controls using Student’s t test. Z p < 0.02 as compared to sham controls using Student’s t test. (6) (4) (6) (5)
0.106 0.187 0.252 0.261
f 5 k +
0.008 0.006’ 0.012* 0.014*
12.50 13.28 14.82 15.66
+ 1 f -c
2,3 Diketoaldonate Decarboxylase
Activity
5.4 5.8 6.4 6.85
+ 0.080 -c 0.063* r 0.090* +- 0.076”
A marked increase of about 60% was observed in the activity of ascorbic acid degrading enzyme, dehydroascorbatase, both in liver and kidney after 12 days of adrenalectomy (Table 3). The increase in the activity of 2,3 diketoaldonate decarboxylase was moderate but highly significant in kidney (Figs. 3 and 4). DISCUSSION
It is apparent from the results that adrenalectomy caused some alteration in the ascorbic acid metabolism in rats. The decrease in the ascorbic acid content of liver in adrenalectomized rats, reported in the present communication, is in accordance with the results of earlier investigators.14J5 Results reported by Shamrai et al.16 are slightly different from those of ours. Their results show that ascorbic acid remained practically unchanged, while dehydroascorbic acid disappeared almost completely and diketogulonic acid increased two to three times in adrenalectomized rats. Dumm and RaliP have reported that adrenalectomy decreases the urinary excretion of ascorbic acid in rats, which is in accordance with our results (Table 1) . Our results also show that adrenalectomy is having certain specific effects on both the biosynthesis and the catabolism of ascorbic acid in rats. A moderate decrease in the formation of ascorbic acid by the total system shows that adrenalectomy diminishes the synthesis of this vitamin in the rat liver. This
Fig. 3.-Effect of adrenalectomy on activity of 2.4 diketoaldonate decarbozylase in liver. A-enzyme activity of shamoperated controls; B, C, D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
f 20e
1042
NATHAN1 ET AL.
Fig. 4.-Effect of adrenalectomy on activity of 2,4 diketoaldonate decarboxylase in kidney. A-enzyme activity of sham-operated controls; B, C, and D: activity of enzyme after 4, 8, and 12 days of adrenalectomy, respectively.
change can be explained on the basis of the decrease found in the activities of ascorbic acid synthesizing enzymes such as b-glucurono-Slactone hydrolase and L-gulono-y-lactone oxidase in rat liver. However, it seems that the catabolism of ascorbic acid is affected most by adrenalectomy (Table 3). There is a marked increase in the activity of dehydroascorbatase both in the liver and the kidney. The other enzyme, 2,3 diketoaldonate decarboxylase, also shows moderate increase. This explains partially, the depletion of dehydroascorbic acid and accumulation of diketogulonic acid in liver of adrenalectomized rats. REFERENCES 1. 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. 2. - and -: The defect in ascorbic synthesis by hypophysectomized rats, Biochem. Biophys. Res. Commun. 4:239, 1961. 3. -, -, and Daginawals, H. F.: Effects of hormones and drugs on activities of Lgulono-y-lactone hydrolase. Proc. Sot. Exp. Biol. Med. 115:800, 1954. 4. Allison, J. E.: Total plasma ascorbic acid as index of stress in rat. Proc. Sot. Exp. Biol. Med. 90:277, 1955. 5. Stewart, C. P., Horn, D. B., and Robson, J. S.: Effect of cortisone and adrenocorticotropic hormones on the dehydroascorbic acid of human plasma. Biochem. J. 53:254, 1953. 6. Sinha, C., and Lahiri, S.: Effect of corticotropin on the concentration of vitamin C in tissues of mice. Indian J. Exp. Biol. 2: 102, 1964. 7. Chelopin, H., Mouton, M., and Ratsimarnanga, A. R.: Some interrelation between ascorbic acid and adrenocortical function. World Rev. Nutr. Diet. 6: 165, 1966. 8. Kagawa, Y.: Enzymatic studies on ascorbic acid catabolism in animals. J. Biothem. (Tokyo) 51:134, 1962.
9. User, B. L.: Hawk’s Physiological Chemistry (ed. 14). New York, McGraw Hill, 1965, p. 1121. 10. Roe, J. H., Mills, M. B., Osterling, M. J., and Damron, C. M.: The determination of Diketo-r_-gulonic acid, dehydro-r. ascorbic acid and L-ascorbic acid in the same tissue extract by the 2,4 dinitrophyenylhydrazine method. J. Biol. Chem. 174:201, 1948. 11. Salomon, L. L., and Stubbs, D. W.: Restoration of aldonolactonase activity by somatotropin in hypophysectomized rats. Biochem. Biophys. Res. Commun. 5:349, 1961. 12. Kagawa, Y., Takiguchi, H., and Shimagono, N.: Enzymatic delactonization of Dehydro-L-ascorbate in animal tissues. Biochim. Biophys. Acta 51:413, 1961. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265, 1951. 14. Giovanni, C., Gerruccio, Di. So, and Andrea, L.: The existence in the rat liver of a probable combined form of vitamin C and its behavior after adrenalectomy. Atti. Sot. Peloritana Sci. Fis. Mat. Natur. 4:289, 1957.
1043
ASCORBIC ACID METABOLISM
15. Cuzzocrea, G., Stefano, F. De., and Lino, A.: Free ascorbic acid in the liver of adrenalectomized rats. Sperimentale 109 : 4 13, 1959. 16. Shamrai, Plattonov, of ascorbic
E. F., Ivanyuta,
0. M.: Contents
0. M., and
of various forms
acid in the tissues of adrenalec-
tomized animals. Ukr. Biokhim. Zh. 37:268, 1965. 17. Dumm, M. E., and Ralli, E. P.: The excretion of pantothenic acid and ascorbic acid by intact and adrenalectomized rats on diets supplemented with and deficient in, pantothenic acid. Endocrinology 45: 188, 1949.