William J. Reddy

Mechanism of action of thyroid hormones on erythrocyte 2,3-diphosphoglyceric acid synthesis

Normal erythrocytes, when incubated with thyroid hormone, were found to have increased levels of 2,3-diphosphoglyceric acid. In addition, a partially purified enzyme preparation, when incubated with either a 1,3-diphosphoglyceric acid generating system or 1,3-diphosphoglyceric acid directly, showed increased levels of 2,3-diphosphoglyceric acid when exposed to thyroid hormone. The hormonal effect was biphasic and was witnessed after 5 min of incubation. Substitution on the 3 and 5 positions of the basic thyronine molecule was essential for hormonal effect. It appears that thyroid hormone acts by directly stimulating the diphosphoglycerate mutase enzyme. The hormonal effect on 2,3-DPG synthesis may offer a biochemical explanation for the shift in the oxyhemoglobin dissociation curve observed in thyroid disorders.

HIGHLY POTENT ADRENAL CORTICAL STEROIDS: STRUCTURE AND BIOLOGIC ACTIVITY

Excerpt Synthetic adrenal steroids possessing biologic activity greater than that of the naturally occurring secretory products of the adrenal cortex are of considerable theoretic importance and, i...

ADRENOCORTICOTROPIC HORMONE IN HUMAN PLASMA

Utilizing the bioassay of Sayers, Sayers and Woodbury (1), based upon adrenal ascorbic acid depletion in the hypophysectomized rat, Paris (2), Taylor (3), and Sydnor (4, 5) and their co-workers, were unable to find detectable levels of ACTH in normal subjects. Gaarenstroom and his colleagues (6) observed detectable quantities in 7 of 33 normal subjects. Others (7-12) have reported very high concentrations. Sayers (13) discounted these latter reports as being inconsistent with the observation (14) that ACTH causes a maximal adrenal response in doses calculated to give much lower plasma concentrations. He concluded that the normal concentration is less than 0.5 mUper 100 ml of blood. Fujita (15) concluded from his studies that the normal concentration of ACTHin whole blood was 1.0 mUper 100 ml. The bioassay, using the secretion of adrenal steroids in the hypophysectomized dog after administration of a test substance (16), provided a more direct method of measuring ACTH, but this method was not sensitive enough to allow measurement of ACTH in the plasma of normal humans. The development of a method for the measurement of corticosterone1 in small quantities of rat plasma (17, 18) permitted the application of thy principle of measurement of adrenal venous corticosteroids to the hypophysectomized rat (19). The rat bioassay for ACTHproved to be sensi-

An assay for adenyl cyclase

Abstract A method for measuring adenyl cyclase in “membrane” fraction of animal tissues has been developed. The assay measures the conversion of 14 C-adenosine 5′-triphosphate to 14 C-adenosine 3′,5′-monophosphate. The incubation is carried out at 30°C in the presence of caffeine, magnesium, phosphoenolpyruvic acid, pyruvate kinase, Tris buffer, and adenosine 5′-triphosphate. The 14 C-adenosine 3′,5′-monophosphate was isolated by paper chromatography and the amount determined by counting in a liquid scintillation counter. The assay has been characterized with respect to optimal pH, magnesium concentration, temperature, caffeine concentration, and time of incubation. Stimulatory effects on the system by sodium fluoride have been observed. The addition of adenosine 3′,5′-monophosphate, guanosine triphosphate, and uridine triphosphate had no effect on adenyl cyclase activity. A linear response with increasing amounts of tissue has been demonstrated. The assay has general applicability to the study of adenyl cyclase in tissues and the effect of various hormones.

Measurement of adenosine 3',5'-monophosphate.

Abstract A double-isotope dilution and derivative analysis technique is described for the measurement of adenosine 3′,5′-monophosphate. Column and paper chromatography are used for the isolation of adenosine 3′,5′-monophosphate from tissues and urine. The nucleotide is esterified with tritium-labeled acetic anhydride via an acetylimidazole intermediate to form adenosine 3′,5′-monophosphate 2′-acetate. Specificity of the technique was demonstrated by repeated chromatography of samples and standards with maintenance of constant specific activity and also by comparison with other techniques using single-isotope dilution analysis. Recovery studies and analysis of proportional quantities of tissues are also presented as evidence of specificity. Technique precision was demonstrated by a coefficient of variation of 7.5% in measurements of 3 × 10 −9 mole of liver adenosine 3′,5′-monophosphate. The practical sensitivity of the technique is 5 × 10 −10 mole. Values for several rat tissues are presented.

Hormonal regulation of myocardial adenosine 3′,5′-monophosphate

Abstract A simple, sensitive, and specific method for measuring changes in adenosine 3′,5′-monophosphate (cyclic AMP) has been applied to the investigation of hormonal control of myocardial cyclic AMP in the rat ventricular slice. Catecholamines have been demonstrated to produce a 100% increase in cyclic AMP levels. Insulin had no effect on cyclic AMP levels and did not alter the catecholamine-induced rise. The presence of Ca2+ in the extracellular fluid was not essential for the production of cyclic AMP, nor was it required for the catecholamine-induced rise in cyclic AMP. Neither glucagon, triiodothyronine or ouabain had any significant effect on cyclic AMP levels in the rat-heart slice.

G-6-PD worcester: A new variant, associated with X-linked optic atrophy

A new variant of glucose-6-phosphate dehydrogenase (G-6-PD), G-6-PD Worcester, is described in a Caucasian family with associated congenital, nonspherocytic hemolytic anemia, absent erythrocyte G-6-PD activity and optic atrophy. The inheritance of this form of optic atrophy is closely linked to the G-6-PD locus and total color blindness. G-6-PD Worcester is distinguished from previously characterized variants by absent erythrocyte G-6-PD activity, slow electrophoretic mobility, thermolability, a sharp peak at pH 8 and a Michaelis constant for TPN of 11.2 micromolar.

BIOLOGICAL EFFECTS OF FLUORINATED DERIVATIVES OF HYDROCORTISONE AND PROGESTERONE IN MAN

The biological effects of halogenated steroids in animals have been reviewed in this monograph by Doctor Fried and his collaborator^.^-^ The high degree of potency which these compounds demonstrated in the animal assays clearly indicated the desirability of a clinical evaluation of their biological activity in I t has been possible to test a number of these derivatives of hydrocortisone and to compare their metabolic effects with those of the nonfluorinated parent compounds. Among them, 9-alpha-fluorohydrocortisone appeared to be the most promising from a clinical point of view and was made available in amounts sufficient for adequate clinical testing. The results obtained with other derivatives are to be considered, at present, as preliminary. These studies have been carried out on the Metabolic Ward of the Peter Bent Brigham Hospital, Boston, Mass. The high degree of activity of 9-alpha-fluorohydrocortisone acetate is illustrated in FIGURE 1. The effects of 100 mg. of hydrocortisone acetate and of 5 mg. of fluorohydrocortisone acetate on urinary electrolyte excretion were compared in an Addisonian patient maintained on a constant diet. The two compounds were administered as a single dose by mouth. I t is apparent that both sodium retention and potassium diuresis were considerably more marked with the fluorinated compound, indicating a t least a twentyfold increase in sodium-retaining activity. Furthermore, when the urinary sodium-to-potassium ratio was followed (FIGURE 2), it appeared that both hydrocortisone and fluorohydrocortisone produced a marked and rapid decrease in this ratio, but that the effects of 5 mg. of fluorohydrocortisone were considerably prolonged as compared to those of 100 mg. of hydrocortisone. The marked effects of 9-alpha-fluorohydrocortisone on organic metabolism are illustrated in FIGURE 3. A patient with Addison’s disease maintained on a constant diet was given 25 mg. of the compound as an eight-hour intravenous infusion. The steroid was dissolved in 10 ml. of absolute ethanol and diluted in 500 ml. of saline containing 5 grams of albumin. The metabolic effects obtained were compared with those of a control infusion of saline with alcohol and albumin. A maximal and prolonged eosinopenia, a marked increase in urinary glucose excretion, and a definite rise in the urinary excretion of nitrogen, uric acid, and potassium were observed with fluorohydrocortisone. Urinary glucose was measured by a specific, enzymatic method based on the oxidation of glucose by glucose oxidase.6 Employing this method with a patient main-

Effect of insulin on the incorporation of [13C]leucine into rat caudofemoralis protein

Abstract This study sought first to develop in in vitro preparation representative of peripheral skeletal muscle and then to study the effects of insulin on the incorporation of labelled amino acid into varous protein fractions thereof. Rat caudofemoralis muscle proteins were separated on the basis of their solubility in solutions of increasing ionic strenght and pH. The specific radioactivity of various protein fractions was found to be inversely proportional to the ionic and alkaline strength of the extracting solution. Insulin stimulated incorporation of [14C]leucine into each fraction with approximately equal magnitude. Likewise, the distribution (ratio) of counts in several protein fractions eluted from a DEAE-cellulose column was not materially affected by insulin. Our results fit the hypothesis that insulin stimulates the incorporation of [14C]leucine into skeletal muscle protein by increasing its entry into cells and not by stimulation of synthesis of one or more specific proteins.

Hormonal effects in vitro on amino acid incorporation into rat adrenal protein: Adrenocorticotrophin and growth hormone☆

Abstract The effects of ACTH and G.H. on glycine-U-C 14 and lysine-U-C 14 incorporation into rat adrenal protein in vitro have been investigated. Increased incorporation was obtained with either ACTH or G.H. but, in combination, there was an inhibitory effect. A dose-response relationship was obtained with both hormones. The effect of either ACTH or G.H. was present in both Krebs-Ringer bicarbonate and phosphate buffer but was inhibited by the presence of 10 m M glucose in the incubation medium. Significant incorporation of amino acids into protein took place after 15 min. of incubation and increased with time up to 1 hr. There was no decrease in incorporation when the glycine concentration was increased 20-fold.