Application of Isotopic Techniques Using Constant Specific Activity or Enrichment to the Study of Carbohydrate Metabolism

ABSTRACT Rat and bovine brain have been incubated with testosterone-4-14C under standard conditions. With use of paper chromatography, the extracted metabolites were noted to fall into less-polar, iso-polar, and more polar fractions. The components of the less-polar fraction were separated by acetylation and thin-layer chromatography and the major end-products identified by recrystallization to constant specific activity or constant 3H/14C ratios. Androst-4-enedione and 5α-dihydrotestosterone were formed consistently under the conditions utilized. Trace amounts of other less-polar metabolites were noted occasionally.

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Effects of insulin on glucose turnover rates in vivo: Isotope dilution versus constant specific activity technique

Metabolism ◽

10.1016/s0026-0495(96)90204-8 ◽

1996 ◽

Vol 45 (1) ◽

pp. 82-91 ◽

Cited By ~ 73

Author(s):

Ole Hother-Nielsen ◽

Jan Erik Henriksen ◽

Jens Juul Holst ◽

Henning Beck-Nielsen

Keyword(s):

Isotope Dilution ◽

Specific Activity ◽

Glucose Turnover ◽

Turnover Rates ◽

Constant Specific Activity

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Theory of production rate calculations in steady and non-steady states and its application to glucose metabolism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1992.262.6.e779 ◽

1992 ◽

Vol 262 (6) ◽

pp. E779-E790 ◽

Cited By ~ 4

Author(s):

J. A. Jacquez

Keyword(s):

Steady State ◽

Experimental Design ◽

Specific Activity ◽

Glucose Production ◽

Central Compartment ◽

Correct Equation ◽

Endogenous Production ◽

Correction Terms ◽

Theory Of Production ◽

Constant Specific Activity

I present a review and synthesis of the basic theory, steady state, and non-steady state for the calculation of metabolite production rates for systems that have a central well-mixed compartment that is the site of tracer input and sampling. The theory is then applied to the calculation of glucose production. If the only inputs are into the central compartment, an experimental design that involves varying tracer infusion rates to maintain constant specific activity in the central compartment and the same constant specific activity in the peripheral compartments allows calculation of the endogenous production. That holds even if the models are unidentifiable. The correct equation and Steele's pool fraction approximation reduce to the same result for this experimental design. However, that does not justify the use of Steele's equation when there are deviations from the exact experimental design. When the specific activity in the central compartment is not constant, model-dependent correction terms to Steele's equation are needed.

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DEFINITIVE IDENTIFICATION OF MICROQUANTITIES OF RADIOACTIVE STEROIDS BY RECRYSTALLIZATION TO CONSTANT SPECIFIC ACTIVITY

Acta Endocrinologica ◽

10.1530/acta.0.049s0007 ◽

1965 ◽

Vol 49 (2_Suppl) ◽

pp. S7-S77 ◽

Cited By ~ 64

Author(s):

Leonard R. Axelrod ◽

Charles Matthijssen ◽

Joseph W. Goldzieher ◽

Jean E. Pulliam

Keyword(s):

Liquid Scintillation ◽

Specific Activity ◽

Inorganic Materials ◽

Dual Isotope ◽

Advantages And Disadvantages ◽

Yield Values ◽

Solvent Systems ◽

Identification Techniques ◽

Constant Specific Activity

ABSTRACT Studies of steroid metabolism using isotopically-labeled compounds at physiological levels present unique problems in the identification of the metabolites and in the demonstration of their radiochemical purity. The submicrogram quantities of material available preclude the use of classical identification techniques. The character of the evidence obtained, the advantages and disadvantages of chromatographic and countercurrent distribution methods are discussed. Crystallization to constant specific activity is a recognized method for demonstrating that a substance is not radiochemically impure. Its parameters have never been accurately defined. Its true power is achieved only when it is preceded by extensive purification of the material to be characterized. In this way, the unknown material is first categorized by its migration rate in various solvent systems, and then by its crystalline identity with the carrier compound. The likelihood of two dissimilar steroids being both isopolar and isomorphic is held to be remote. Liquid scintillation spectrometry and gravimetry are the techniques used for the determination of constant specific activity. This method for measurement of radioactivity is extremely flexible, sensitive, and lends itself to dual-isotope experiments. Gravimetry under standardized conditions is suitably precise and much more generally applicable than spectroscopic quantitation. The parameters of the technique of rapid, forced microcrystallization are analyzed. In particular, the problem of contamination of crystals is analyzed in detail, and it is pointed out that classical concepts of purification by crystallization, developed chiefly in connection with ionic inorganic materials, must be modified when applied to nonionic steroid compounds. A mathematical analysis of the errors inherent in this technique indicates that 3 successive crystallizations of a pure radioactive compound should yield values for the specific activity which are within ± 5 % of the average of the 3 values.

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Preparation of 14C- and 3H-Labeled Aflatoxins

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/54.5.1027 ◽

1971 ◽

Vol 54 (5) ◽

pp. 1027-1031

Author(s):

J L Ayres ◽

D J Lee ◽

R O Sinnhuber

Keyword(s):

Thin Layer ◽

Aspergillus Flavus ◽

Column Chromatography ◽

Aflatoxin B1 ◽

Sodium Acetate ◽

Specific Activity ◽

Tritiated Water ◽

New Method ◽

Extraction And Purification ◽

Constant Specific Activity

Abstract A new method for the preparation of 14C- and 3H-labeled aflatoxins was devised, using rice as a supporting mold media. Labeled precursors were added to sterile rice and the mixture was inoculated with Aspergillus flavus spores. After a 7 day incubation at 25°C, the toxins were extracted with chloroform and purified by column chromatography and subsequent recrystallization. Aflatoxins B1 and G1 were recovered with 70% efficiency from the culture. Incorporation of radioactivity was examined with glucose-U-14C, sodium acetate-1-14C, and sodium acetate-2-14C. The latter gave the most efficient incorporation of 14C at 0.1% for aflatoxin B1 and 0.05% for aflatoxin G1. Conversion of 3H from tritiated water was 0.006% for aflatoxin B1 and 0.003% for aflatoxin G1. Extensive tests of radiopurity were performed on the labeled toxin which included: recrystallization to constant specific activity, thin layer and column chromatography, and hydrogenation of aflatoxin B1 to tetrahydrodeoxoaflatoxin B1. The rice-culturing technique gave good toxin yields of 1 mg aflatoxin B1/g rice. The purification was simplified by the absence of highly radioactive impurities and no appreciable degradation of labeled toxins was noted throughout extraction and purification.

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Carbohydrate Metabolism of Hypophysectomized Dogs as Studied With Radioactive Glucose

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1956.187.1.25 ◽

1956 ◽

Vol 187 (1) ◽

pp. 25-31 ◽

Cited By ~ 28

Author(s):

R. Steele ◽

J. S. Wall ◽

R. C. de Bodo ◽

N. Altszuler

Keyword(s):

Body Weight ◽

Carbohydrate Metabolism ◽

Surface Area ◽

Body Surface Area ◽

Body Surface ◽

Specific Activity ◽

Constant Infusion ◽

Minute Period ◽

Endogenous Glucose ◽

Low Rate

Minute amounts of uniformly labeled C14 glucose were administered intravenously to unanesthetized normal and hypophysectomized dogs, in the postabsorptive state, as an initial priming injection, followed by a constant infusion. From the observed specific activity of the plasma glucose during the 60–180-minute period of the constant infusion, the following parameters of carbohydrate metabolism were determined: a) the size of the glucose pool, b) the glucose space and c) the rate of turnover of the glucose pool. The rate of total CO2 production was also determined. The rate of total CO2 production, per square meter of body surface area, was found to be less in the hypophysectomized dog than in the normal one. The glucose pool, per kilogram body weight, was found to be smaller in the hypophysectomized dog than in the normal one. The glucose space, expressed as percentage of body weight, was found to be similar in the two types of animals. The rate of turnover of the glucose pool in the hypophysectomized dog, presented as grams glucose per square meter of body surface area per hour, was found to be less in the hypophysectomized dog than in the normal one. The low rate of glucose uptake by the tissues which was observed to prevail in the hypophysectomized dog is believed to reflect an adjustment in the secretion of insulin to conform to the limited availability of endogenous glucose which results from the removal of the pituitary gland.

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THE INFLUENCE OF CHANGING CELL KINETICS ON FUNCTIONAL DIFFERENTIATION IN THE SMALL INTESTINE OF THE RAT A STUDY OF ENZYMES INVOLVED IN CARBOHYDRATE METABOLISM

Journal of Histochemistry & Cytochemistry ◽

10.1177/22.5.352 ◽

1974 ◽

Vol 22 (5) ◽

pp. 352-360 ◽

Cited By ~ 21

Author(s):

N. J. DE BOTH ◽

H. PLAISIER

Keyword(s):

Carbohydrate Metabolism ◽

Enzyme Activities ◽

Cell Kinetics ◽

Specific Activity ◽

Functional Differentiation ◽

Radiation Doses ◽

Germ Free ◽

Cell Age ◽

Glucose 6 Phosphate Dehydrogenase ◽

Low Radiation Doses

The activity of a number of enzymes involved in carbohydrate metabolism was measured in different cellular compartments of the intestinal epithelium by microchemical techniques. The enzyme activities were related to different cell positions along crypt and villus and to cell age. Enzyme activities in proliferating, differentiating and functional cell compartments of the intestine of normal rats were compared with those in which the cell kinetics had been modified. The effect of increased proliferative activity within the crypt of normal animals was studied in the intestine during recovery after low radiation doses. The effect of increased life-span was investigated in germ-free animals. The specific activity of α-glucosidase, present in microvilli, was found to increase considerably during cell differentiation and subsequent cell migration along the villus. Its specific activity remained unchanged in isolated intestinal loops deprived of dissaccharide substrate for 6 weeks. Lactate dehydrogenase and, to a lesser extent, glucose 6-phosphate dehydrogenase show a similar pattern of activity to α-glucosidase. In contrast hexokinase and isocitrate dehydrogenase were equally distributed among crypt and villus cells and no clear differences were observed with increasing cell age. Increased proliferation in the crypts during recovery after low radiation doses resulted in a marked decrease in both crypt and villus cells of activity of α-glucosidase. The germ-free state of the intestine also significantly influences the pattern of α-glucosidase but has little influence on the other enzymes tested.

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GLUCOCORTICOID SYNTHESIS FROM PREGNENOLONE BY SHEEP FOETAL ADRENALS IN VITRO

Journal of Endocrinology ◽

10.1677/joe.0.0580485 ◽

1973 ◽

Vol 58 (3) ◽

pp. 485-491 ◽

Cited By ~ 9

Author(s):

I. J. DAVIES ◽

K. J. RYAN

Keyword(s):

Isotope Dilution ◽

Adrenal Glands ◽

Specific Activity ◽

Newborn Period ◽

Adult Sheep ◽

Age Related ◽

Period Of Gestation ◽

Constant Specific Activity

SUMMARY [7-3H]Pregnenolone was incubated with homogenates of adrenal glands from two 100-day-old sheep foetuses. Cortisol and corticosterone were isolated and identified by reverse isotope dilution and recrystallization to constant specific activity. Together these two compounds accounted for 12% and 17% of the substrate with the two tissue preparations. Other C21 and C19 metabolites which were sought were not present in appreciable quantities. Additional incubations were done with the adrenals of lamb foetuses ranging in age from 110 days of gestation to the immediate newborn period. Glucocorticoidogenic capacity similar to that of the 100-day-old foetuses was demonstrated throughout this period and no age-related change was evident. These results demonstrate that the lamb foetal adrenal has a substantial enzymic capacity for glucocorticoid synthesis throughout at least the last third of gestation. In conjunction with the observations of others, these experiments support the hypothesis that during this period of gestation the lamb foetal adrenal is actively synthesizing glucocorticoids in a manner which is similar to the lamb at term and the adult sheep.

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FORMATION OF 17α-HYDROXY-PREGNENOLONE, 17α-HYDROXY-PROGESTERONE AND PROGESTERONE BY HYDATIDIFORM MOLES IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.0610068 ◽

1969 ◽

Vol 61 (1) ◽

pp. 68-75 ◽

Cited By ~ 2

Author(s):

Hubertus A. van Leusden ◽

Maria Siemerink

Keyword(s):

Urinary Excretion ◽

Specific Activity ◽

Full Term ◽

Hydroxy Progesterone ◽

Experimental Findings ◽

Hydatidiform Moles ◽

Constant Specific Activity

ABSTRACT Vesicles of hydatidiform moles were incubated in the presence of [7α-3H]pregnenolone, After the incubation and extraction of tissues and media, 17α-hydroxy-pregnenolone*, 17α-hydroxy-progesterone and progesterone were identified using a number of TLC systems, followed by crystallization to a constant specific activity. [7α-3H] pregnenolone was not converted to oestrone, 17β-oestradiol and oestriol. The experimental findings indicate that hydatidiform moles, like full term placentas, are deficient in the enzymes necessary to convert C21 to C19 steroids. The production of 17α-hydroxy-progesterone and progesterone in the molar trophoblast in situ may contribute to the considerable urinary excretion of pregnanetriol and pregnanediol in patients with hydatidiform moles.

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Effects of 5-hydroxytryptamine on carbohydrate metabolism in Hymenolepis diminuta (Cestoda)

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y83-020 ◽

1983 ◽

Vol 61 (2) ◽

pp. 137-143 ◽

Cited By ~ 15

Author(s):

M. S. Rahman ◽

D. F. Mettrick ◽

R. B. Podesta

Keyword(s):

Carbohydrate Metabolism ◽

Gas Phase ◽

Specific Activity ◽

Energy Charge ◽

Hymenolepis Diminuta ◽

Total Carbon ◽

Adenylate Energy Charge ◽

Acetate Excretion ◽

Carbon Transfer ◽

Adenine Nucleotide Pool

Under in vitro anaerobic and aerobic incubations the presence of 5-hydroxytryptamine (5-HT) resulted in an accelerated rate of carbon transfer through the carbohydrate metabolic pathways of Hymenolepis diminuta resulting in an increase in worm tissue total carbon pools. Under aerobic incubation, levels of phosphoglycerates, phosphoenolpyruvate (PEP), and pyruvate were significantly increased over the corresponding levels under anaerobic incubation. Excreted end products of carbohydrate metabolism also differed significantly depending on the gas phase and the presence of 5-HT. Under atmospheric (21%) oxygen concentrations excreted levels of lactate, succinate, and acetate were all significantly elevated; under a 95% O2–5% CO2 gas phase only lactate excretion was increased, and under a 95% N2–5% CO2 gas phase only succinate and acetate excretion increased. The presence of oxygen reduced acetate excretion by up to 55% and under the 95% O2–5% CO2 gas phase succinate excretion was reduced 48%. Irrespective of gas phase or the presence of 5-HT, adenine nucleotide pool sizes and the adenylate energy charge remained constant. The addition of 5-HT had no significant effect in increasing the specific activity of phosphofructokinase (PFK). In the presence of ATP the addition of cAMP increased PFK activity by up to 48%; AMP enhanced the enzyme activity by up to 69%, irrespective of the presence or absence of 5-HT. These results are discussed in terms of the effects of 5-HT, gas phase, and nucleotide pools on the motility, metabolism, and control of H. diminuta, and of other parasites which respond to 5-HT.

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