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

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.

Constant specific activity input allows reconstruction of endogenous glucose concentration in non-steady state

In vivo studies on the glucose system often require its perturbation by an exogenous input of glucose, whereas glucose turnover is assessed by infusing a glucose tracer. The constant infusion represents the usual format of tracer administration, but it has no clear advantage other than simplicity. Here we propose a different tracer infusion format. It consists of infusing the tracer in parallel with unlabeled glucose so as to maintain a constant specific activity in the infusate. This protocol does not increase experimental complexity and provides new information on the glucose system in non-steady state by allowing reconstruction of the endogenous component of glucose concentration. This reconstruction only requires very general assumptions, such as tracer-tracee indistinguishability and mass conservation; in particular it is independent of the glucose model structure, i.e., number of compartments and their interconnections. A proof of the result is given for a general nonlinear model of the glucose system. The constant specific activity input is also advantageous for non-steady-state calculations, because it reduces the variation in the measured plasma glucose specific activity. The glucose system has served as the prototype, but the protocol is applicable to other blood-borne substances. The radioactive tracer case has been considered, but the same results apply to stable isotope tracers as well; in this case they also become relevant in a somewhat different context, i.e., kinetic studies in steady state.

Stimulation of aromatase activity in the fetal rat gonads by cAMP and FSH

The gonads from 17- to 21-day-old fetal rats were cultured in vitro in the presence of [3H]testosterone and in the presence or absence of cAMP or FSH, and estrone and estradiol formed were measured by double isotopic dilution and recrystallization to constant specific activity. Estrogen synthesis by testes was stimulated by both cAMP and FSH as early as at 18 days of gestation. FSH did not enhance aromatase activity in ovaries, although cAMP did. It is remarkable that FSH controls estrogen synthesis in the testis earlier than in the ovary.

IDENTIFICATION OF STEROIDS PRODUCED BY BOVINE CHORION INCUBATED WITH [14C]ANDROSTENEDIONE

Individual chorion taken from cows in early pregnancy (days 27, 44, 45 and 49) was incubated with [14C]androstenedione in short-term incubation in vitro. After preliminary extraction, separation and purification, metabolites were identified by recrystallization with authentic unlabelled steroids to constant specific activity. Major metabolites identified were 5β-androstane-3,17-dione (5β-androstanedione) and 5β-androstan-3α-ol-17-one (aetiocholanolone) while minor metabolites were identified as 5β-androstane-3α,17β-diol and 5β-androstan-17α-ol-3-one. There was no 14C-labelled oestradiol or oestrone detectable in the medium either as free oestrogens or as sulphates.

Corrigendum

In the paper by Charlotte A. Llewelyn (J. Endocr. 1981, 89, 283–288) the first two sentences of the Results section should read as follows. The area on the initial chromatogram run corresponding to progesterone contained one 14C-labelled peak coincident with carrier progesterone in solvent systems 4, 5 and 6. The area corresponding to androstenedione had the same relative front (RF) value as carrier androstenedione in solvent systems 3, 4 and 6 and was recrystallized to constant specific activity, e.g. after four successive recrystallizations the percentage change in radioactivity per mg was 1·2 ± 1·6 (mean ± s.e.m.).

The Distribution of Photoassimilated Carbon and the Growth of Douglas-fir Seedlings

Two-year-old Douglas-fir seedlings accumulated photoassimilated carbon in the tissues of new needles, previous years' needles, new shoots, stem, and roots at rates relative to tissue carbon of 1.25, 0.57, 0.42, 0.60, 0.61%/d, respectively. These daily net accumulation rates were measured just after bud set, and the rates parallel the carbon accumulation expected based on phenological observation. Results were obtained with a new system for long-term labelling of groups of seedlings with 14CO2 at a constant specific activity. This system is described.

GLUCOCORTICOID SYNTHESIS FROM PREGNENOLONE BY SHEEP FOETAL ADRENALS IN VITRO

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.