Adrenal Ascorbic Acid Depletion Research Articles

The corticotrophic activity of tetracosactide in the adrenal ascorbic acid depletion test.

Abstract Using the subcutaneous adrenal ascorbic acid depletion test in hypophysectomized rats it was found that tetracosactide (synthetic 1–24 corticotrophin) could not be assayed against the International Working Standard (IWS) for natural corticotrophin because of a difference in the slopes of the dose-response curves of the preparations. Valid potency estimates were obtained when tetracosactide was assayed against a standard preparation closely similar to the 2nd International Standard for natural corticotrophin. By comparing this standard with the IWS, the best estimate of the potency of tetracosactide in terms of the IWS was 99 (92–106) units/mg. Five different batches of tetracosactide have yielded identical biological potencies. Since tetracosactide can be adequately standardized by physical and chemical-analytical criteria, labelling and dosage on a mg base instead of in units is recommended.

Inhibition of spermatogenesis in house rat (Rattus rattus Rufescens) following the administration of alpha- chlorohydrin.

Chronic administration of alpha-chlorohydrin (25 mg/kg body wt.) caused lesions in the testis of house rat. The changes in the germ cells were degenerative. The seminiferous tubules and Leydig cell nuclear diameter were reduced. Epididymal cell height was greatly reduced. The lumen was devoid of spermatozoa. alpha-Chlorohydrin administration inhibited the synthesis of RNA, protein, sialic acid in the testis and accessory sex organs. Total cholesterol of the testis was increased. Adrenal ascorbic acid depletion was conspicuous. alpha-Chlorohydrin anatagonizes the stimulatory action of testosterone in castrated house rats. alpha-Chlorohydrin can be considered as a biologically effective compound for the control of house rat population in human dwellings. It's dose dependent reaction is discussed.

Systemic administration of adenosine ameliorates pentylenetetrazol‐induced chemical kindling and secondary behavioural and biochemical changes in mice

Adenosine is one of the inhibitory neuromodulators in the brain and is considered to be responsible for seizure arrest and postictal refractoriness. Adenosine, adenosine receptor agonists, and adenosine uptake blockers are known to reduce the severity and duration of amygdala-kindled seizures. The present study was carried out to elucidate the anticonvulsant and neuromodulatory effect of systemic adenosine on the pentylenetetrazol (PTZ)-induced chemical kindling in mice. Kindling was induced by chronic administration of a subconvulsive dose of PTZ (40 mg/kg, i.p.) on every other day for a total period of 9 days. Adenosine was administered daily, 30 min before PTZ or vehicle. The kindling score was recorded immediately following PTZ administration according to a prevalidated scoring scale. Various behavioral and biochemical estimations were performed on day 10 (i.e. 24 h after the last dose of PTZ). Chronic PTZ treatment progressively increased the seizure score with the maximum score reached on day 9. Behavioral analysis found hyperlocomotor activity, anxiogenic response, hyperalgesia and amnesia in kindled mice. Biochemical analysis revealed that chronic treatment with PTZ significantly increased lipid peroxidation (malondialdehyde levels), nitrite (NO(2-) levels), adenosine deaminase (ADA) and total RNA levels and decreased catalase, reduced glutathione (GSH) levels in brain homogenates, and a depletion of adrenal ascorbic acid. Daily treatment with adenosine (25 and 50 mg/kg, i.p.) for 9 days led to a significant decrease in PTZ-induced kindling score and also reversed various behavioral and biochemical alterations produced by PTZ. The results of the present study suggested that systemic adenosine administration reversed the behavioral and biochemical alterations induced by chronic PTZ.

Adrenal ascorbic acid depletion as an index of preslaughter stress in pigs

The weight of the adrenal glands and the amount of ascorbic acid they contained were measured in Large White pigs subjected to various preslaughter treatments. Compared with transport for four hours preslaughter, one hour's transport resulted in adrenal glands which weighed less and which contained less ascorbic acid. Pigs mixed with strange pigs in lairage had heavier adrenals containing more ascorbic acid than those of unmixed pigs. Increasing lairage time up to twenty-four hours raised both the concentration and total amounts of ascorbic acid in the adrenal glands. The results suggest that the depletion of ascorbic acid from the adrenal glands can be used as a measure of the stress experienced by animals during the preslaughter period and the extent to which resting in lairage allows them to recover.

Liver tyrosine-alpha-ketoglutarate transaminase as a quantitative test of the phlogistic potency of agents topically applied

The liver tyrosine-alpha-keto-glutarate transaminase (TKT) increase, provoked in rats by topical application on the conjunctiva of various phlogistic agents such as croton oil, mustard oil, formaldehyde and veratrine sulphate, is shown to be available as test for a quantitative evaluation of the so elicited inflammation. In fact, the TKT increase (measured at the 150th min after the application) is linearly related to the log of increasing concentration of the tested drugs and the parallelism of the calculated regression lines enables to calculate the potency of various agents with reference to croton oil, the comparison of the test with the adrenal ascorbic acid (AAA) depletion test, simultaneously evaluated, shows a substantial agreement of the values of the potencies and their precision. On the basis of the well-known relationship between glucocorticoid secretion and AAA depletion and TKT increase, the hypothesis that any phlogistic drug stimulates the hypothalamus pituitary-adrenal axis isconfirmed.

Inhibition of Huggins tumors by forced restraint.

Three experiments demonstrated that chronically administered restraint inhibits the development of DMBA-induced rat mammary tumors. The effect is exhibited in a lower proportion of positive responses, increased latency of tumor development, and lower number of tumors in positive animals when restrained animals are compared with controls. Organ weights failed to show a stress response to restraint. Molar activity data from Experiment III indicated that early in the experiment activity increased among the restrained animals on release from restraint. A fourth experiment, which employed a crystal accelerometer to assess activity, produced a similar activity pattern and also provided evidence of adrenal ascorbic acid depletion as a function of restraint.

Quantitative evaluation of antiphlogistic effect of corticosteroids topically applied

Summary The adrenal ascorbic acid depletion, elicited in rats by conjunctival phlogosis provoked by application of croton oil, has been assumed as a test for quantitative evaluation of antiphlogistic effect of the following topically applied steroids: hydrocortisone acetate, triamcinolone acetonide, dexamethasone valerate. It is shown that the ascorbic acid depletion is reduced by such steroids and that such a reduction is linearly related to the log of their increasing concentrations. The parallelism of the calculated regression lines enables the evaluation of their antiphlogistic potency in reference to hydrocortisone acetate, which is 3.80 for triamcinolone and 4.27 for dexamethasone. The results obtained are compared with those obtained by the other experimental methods devised in order to evaluate the topical antiphlogistic effect of corticosteroids.

Plasma corticosterone in rats after electrochemical stimulation of the olfactory bulbs.

The effect of stimulation and lesions of the extrahypothalamic neural structures on corticotrophin (ACTH) secretion has been widely investigated (Mangili, Motta & Martini, 1966; Rubin, Mandell & Crandall, 1966; Redgate, 1970). Palma, Perassi & Loyber (1971) found that electrochemical stimulation of the olfactory bulbs in rats produced depletion of adrenal ascorbic acid, while their resection (Loyber, Lecuona, Palma & Perassi, 1971) decreased the urinary 17-oxosteroids. Plasma corticosterone levels were therefore investigated in rats to determine the effect of olfactory bulb stimulation on ACTH secretion. White, female, virgin rats (150–180 g) bred in our institute were used. They were all kept under the same conditions and divided into three groups subjected to the following: (a) bilateral electrochemical stimulation of olfactory bulbs; (b) bilateral electrochemical stimulation of parietal cerebral cortex; (c) sham stimulation of olfactory bulbs. Electrochemical stimulation was effected by a stainless steel electrode, 0·3 mm diameter, passing 1 mA, d.c.

Role of adrenal glands in the serum protein changes produced by resection or stimulation of the olfactory bulbs in the rat

The role of the adrenal glands on the serum protein changes produced by resection or stimulation of the olfactory bulbs of the rats was studied. The production of an increase of albumin and alpha globulin by the stimulation of the olfactory bulbs requires the presence of the adrenal glands. The depletion of adrenal ascorbic acid observed after the stimulation of the olfactory bulbs indicates an increase of the liberation of ACTH. The normalization of albumin and alpha globulin, produced by adrenal substitutive therapy in bulbectomized rats indicates that the decrease of these fractions is produced by adrenal insufficiency.

Synthesis of corticotropin peptides. XII. The effect of amino-terminal substitution in the corticotropin 1-18 peptide.

Abstract The synthesis and biological properties are described of an octadecapeptide amide, α-aminoisobutyryl-tyrosyl-seryl-methionyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyl-glycyl-lysylprolyl-valyl-glycyl-lysyl-lysyl-arginyl-arginine amide, corresponding to the first eighteen amino acid residues of corticotropin (ACTH) except for the amino terminal residue. The amino-terminal decapeptide as intermediate is built up from a tripeptide (positions 1–3) and a heptapeptide (4–10), where a benzyloxycarbonyl and a benzyl ester groups are used for protection of the terminal α-amino group and the γ-carboxyl group of 5-glutamic acid, respectively. These protecting groups are removed in the final step with hydrogen fluoride. The new method is successfully applied to the corresponding 1-β-alanine octadecapeptide amide, which has been synthesized previously by a different procedure. Adrenal-stimulating properties of the 1-α-aminoisobutyric acid peptide are compared with those of the 1-β-alanine peptide to show that the former peptide is more active than the latter when assayed by the adrenal-ascorbic acid depletion and the in vivo steroidogenesis methods. The lipotropic activity of the new peptide is also shown to be more prolonged than that of the 1-β-alanine peptide.

A study of ACTH and CRF in chickens

Previous work had demonstrated that chicken pituitaries contained a substance which was able to cause corticosterone synthesis by adrenals in vitro. In the present study chicken pituitary glands were assayed by the adrenal ascorbic acid-depletion method in hypophysectomized rats. It was found that perfect parallelism existed between the doses of 1.5–96.0 μg/100g body weight of chicken adenohypophyses and 0.4–10.0 mU of a standard mammalian ACTH. The data presented show that chicken pituitary glands contain relatively more ACTH than do those of rats. When assayed on hypophysectomized rats, perfect parallelism was demonstrated between extracts of 10, 20, and 40 chicken hypothalamic fragments and 0.4, 2.0, and 10.0 mU of the ACTH standard. Similar data were obtained on intact blocked rats. These results led to the conclusion that chicken hypothalami contain ACTH or an ACTH-like substance. No activity was present in extracts of chicken cerebral cortices which were used as control test substances.

Corticosteroid response to natural and synthetic corticotrophins in the autotransplanted sheep adrenal.

Third International Standard ACTH was compared quantitatively with synthetic β1-24-corticotrophin by an intravenous method which used sheep with isolated adrenal glands autotransplanted to a vascular anastomosis in the neck. The 2 corticotrophin preparations, which had been standardized by the adrenal ascorbic acid depletion test, were found by this method to be closely comparable. The potentialities of this experimental situation as a basis for a bioassay for ACTH are discussed. The bioassay was found to have a minimum effective dose of 0.02 mU Third International Standard ACTH, or 0.0002 Mg of synthetic β1-24 corticotrophin. (Endocrinology 85: 570, 1969)

Effects of social stimuli on the adrenal cortex in male mice

Male mice were housed either in wire-mesh cages placed adjacent to one another or in similar cages separated by wooden partitions. A higher level of adrenocortical function, measured by relative adrenal weights and adrenal ascorbic acid depletion, was found in the mice which had been allowed partial social contact. These results demonstrate that the adrenal cortex not only responds to social stimuli traditionally regarded as stressors but is also sensitive to more subtle changes in the social invironment.

Distribution of ACTH in the hypophysis of necturus

AbstractThe winter Necturus hypophysis both whole and selectively divided was extracted in N/10 HCl (0.25 ml per gland or gland portion). Extracts were diluted with normal saline (1:100 and 1:400) and injected 2 ml/100 gm body weight) into groups of 10 or 12 male, hydrocortisone‐blocked assay rats (method of Hodges and Vernikos ('60). Groups of control hydrocortisone‐blocked male rats received corresponding volumes of a standard ACTH solution. ACTH content of extracts was appraised by measuring the adrenal ascorbic acid depletion induced as compared with control depletions. A 2 + 2 assay design was employed; results and their fiducial limits (P = 0.95) calculated.The ACTH content of the anterior zone of pars buccalis is that of the entire hypophysis. No ACTH was detected in gland portions consisting of the basophilic bed and transitional zone of pars buccalis plus the neural lobe of pars neuralis. ACTH occurs throughout the anterior zone but is more abundant in its caudal two‐thirds both in milliunits (mU) per milliliter of extract and mU/mg of pituitary tissue. This distribution of corticotrophic activity is coincident with the distribution of anterior zone acidophile cells.Regional and cellular sources of ACTH in mammals and amphibia are discussed.

Depletion of ascorbic acid from the adrenal of the intact embryo of gallus domesticus by adrenocorticotrophic hormone or histamine

1. 1. In four our five experiments ACTH (45 i.u./kg, i.v.) caused a significant depletion of adrenal ascorbic acid withing 5 min. Repletion began after 30 min and was substantially complete within 30 min. In the fifth experiment, however, there was an immediate and a significant increase in the ascorbic acid level. 2. 2. Histamine (3 mg/kg, i.v.) caused a transient but significant rise in adrenal ascorbic acid. After 10 min there was a significant fall. Repletion was advanced 1 hr after stimulation. 3. 3. The possible role of the bursa of Fabricius in adrenal ascorbic acid metabolism is discussed in the light of these results.

Ascorbic acid depletion and corticosterone production in the avian adrenal gland

Adult White Leghorn hens maintained under a 13-hour light day (7 am to 8 pm) and killed between 8:00 and 10:30 am exhibited no adrenal ascorbic acid depletion following the administration of ACTH. Treatment with pharmacological doses (100 mg) of cortisol produced a significant lowering of adrenal ascorbic acid levels while much lower doses of the hormone (2.5 mg/kg body weight) induced a rise in the ascorbic acid level. The injection of ACTH in a group of birds pretreated with 2.5 mg of cortisol/kg body weight produced a significant fall in the adrenal ascorbic acid. The effects of various experimental factors on in vitro adrenal corticosterone production were also investigated. Either the addition of ACTH to the incubation medium or pretreatment of hens with ACTH greatly stimulated corticosterone production by avian adrenal slices. Pretreatment of adult hens with either low or pharmacological doses of cortisol depressed the in vitro production of corticosterone and depressed the responsiveness of the adrenal gland to exogenous ACTH.

Effect of stress on the ascorbic acid content of the adrenal gland of Gallus domesticus

1. 1. There is a significant depletion of ascorbic acid from the adrenal gland within 10 min of handling a group of young chickens. 2. 2. Despite further stimulation, rapid repletion occurs after this time and is substantially complete 25 min after the initial stimulation. 3. 3. The hypothesis is advanced that the bird responds to stress in a similar way to the mammal but that the response, as measured by adrenal ascorbic acid depletion, is considerably more rapid.

Long-term effects of intracerebral corticoid implants.

ABSTRACT Chronic implantation of cortisol acetate in the basal medial hypothalamus resulted in a steady decrease in weight of the adrenal glands which remained severely atrophic up to 70 days post-implantation. At this time, however, the adrenal ascorbic acid depletion response to unilateral adrenalectomy was normal. The compensatory adrenal hypertrophy (CAH) response, which was inhibited in the immediate post-operative period, reappeared later, and had returned to normal by 21 days postoperatively. Intramuscular administration of cortisol in unimplanted rats inhibited CAH at 14 or 21 days following onset of treatment, and prevented the recovery of CAH in animals implanted 21 days previously with cortisol in the median eminence. The possibility is discussed that the differential recovery of the responses to unilateral adrenalectomy in implanted animals with continuing atrophy of the adrenal cortex is due to some adaptation of central nervous mechanisms subserving the CAH response.

ON THE PRESENCE OF PRECORTICOTROPHIN – A PRECURSOR OF CORTICOTROPHIN IN VARIOUS MAMMALIAN HYPOPHYSES

ABSTRACT Pituitary extracts of various species origin such as beef, sheep, pig, guineapig, rabbit and rat prepared in 0.9 per cent sodium chloride solution were assayed for corticotrophic activity, before and after acid treatment of the samples, by the adrenal ascorbic acid depletion method. The corticotrophic activity per mg of the pituitary tissue of sheep, pig and guineapig is comparable, but is considerably less than that of rat and rabbit pituitary glands. At the dose level of 0.25 mg wet weight of pituitary tissue per 100 g of body weight of the assay animals, the pituitary glands of all species except rabbit did not show any corticotrophic activity before acid treatment. However, after acid activation of the preparations significantly higher corticotrophic activity was detectable at this dose level. It is concluded that (1) the presence of precorticotrophin in the mammalian adenohypophysis is a general phenomenon, (2) at least 80 per cent of the total corticotrophic activity of the pituitary glands exists in the form of precorticotrophin, and (3) pig pituitary gland would be a suitable starting material for the separation of precorticotrophin from other pituitary factors. Cold stress caused substantial depletion in the pituitary glands of both corticotrophin and precorticotrophin. Corticosterone treatment and adrenalectomy caused a profound decrease in the corticotrophin content of the hypophysis but precorticotrophin was clearly demonstrable.

Comparison of plasma corticotropin concentration in intact and adrenalectomized dogs and cats during hemorrhage.

The concentration of ACTH in arterial blood samples from dogs and cats was determined by the adrenal ascorbic acid depletion assay or the plasma corticosterone assay. ACTH was extracted from plasma samples by running acidified plasma through IRC-50 resin columns. The mean recovery of endogenous ACTH in the eluate from these columns was 80%. The mean recovery of exogenous ACTH added to dog blood was 91%. The increase in plasma ACTH concentration induced by hemorrhage in dogs and cats at 2 hr after adrenalectomy was greater than the increase in concentration observed in shamadrenalectomized and intact animals. This marked augmentation of the response to hemorrhage after adrenalectomy suggests that the 80–90% decrease in circulating corticosteroids known to occur in the 2 hr after adrenalectomy is sufficient to increase the rate of release of ACTH during stress. Similar results were obtained when adrenal cortical secretion was reduced by the administration of metyrapone (Su-4885). (Endocrinology 80: 741, 1967)