R. De Hertogh

Circadian variations of blood sugar and plasma insulin levels in man.

SummaryBlood sugar, plasma insulin, non-esterified fatty acids (NEFA), plasma cortisol, and urinary catecholamines were measured for 24 h in seven normal subjects receiving a standard diet. During the night, blood sugar and plasma insulin remained low, NEFA decreased progressively, and the excretion of catecholamines diminished. During the day, the insulin response appeared particularly important after the morning meal. This last observation was also made when normal subjects were given three identical meals at intervals of four and a half hours. Under these conditions, the postprandial elevations of blood sugar were not statistically different, but the plasma insulin rose significantly higher after the morning meal. These observations may be explained by the existence of a periodicity which would regulate the insulin secretion. It is also possible that the insulin liberated postprandially conserves a certain activity at the moment of the next meal, and still intervenes in the maintaining of blood sugar homeostasis. Later in the day, however, blood sugar homeostasis would necessitate a new synthesis of insulin, which would explain the delayed plasma insulin response to the evening meal.RésuméLa glycémie, rinsulinémie, les acides gras non estérifiés, le cortisol plasmatique ainsi que les catecholamines urinaires ont été mesurés pendant 24 h chez sept sujets normaux recevant une alimentation standardisée. Durant la nuit, la glycémie et linsulinémie restent basses, les taux plasmatiques dacides gras non estériflés diminuent progressivement et les catecholamines sont excrétées en moins grande quantité. Pendant le jour, la libération dinsuline paraît particulièrement importante après le repas du matin. Cette dernière constatation se vérifie aussi lorsquon administre à des sujets normaux trois repas identiques distants de quatre heures et demie. Dans ces conditions, en effet, les augmentations postprandiales de la glycémie ne sont pas statistiquement différentes, mais laccroissement de linsulinémie est significativement plus important après le repas du matin. Ces observations peuvent sexpliquer par lexistence dune périodicité qui régirait linsulino-sécrétion. Il est aussi possible que linsuline libérée après un repas conserve une certaine activité et intervienne encore dans le maintien de lhoméostasie glycémique au moment du repas suivant. Le soir, laugmentation prolongée de linsulinémie suggère lexistence dune nouvelle synthèse dinsuline.ZusammenfassungBei sieben normalen und zwei übergewichtigen Personen, die standardisierte Mahlzeiten einnahmen, wurden während der Zeitspanne von 24 h Blutzucker, Plasma-Insulin, freie Fettsäuren, Plasma-Cortisol sowie die Katecholamine im Urin bestimmt. Nachts bleiben Blutzuckerspiegel sowie Insulinkonzentration relativ niedrig, nehmen die freien Fettsäuren progressiv ab und verringert sich ebenfalls die Katecholamin-Ausscheidung. Tagsüber ist die Insulinfreisetzung nach der Morgenmahlzeit stärker als mittags und abends. Dies trifft gleichfalls zu, wenn normalen Personen dreimal die gleiche Mahlzeit nach jeweils 4 1/2 h verabreicht wird. Unter diesen Bedingungen ergibt sich, daß die Steigerung des Blutzuckerspiegels nach den jeweiligen Mahlzeiten nicht statistisch verschieden ist, jedoch der Insulinspiegel nach der Morgenmahlzeit signifikant höher liegt. Diese Beobachtungen können durch die Existenz einer Periodizität der Insulinsekretion zu erklären sein. Es ist ebenfalls möglich, daß das nach einer Mahlzeit sezernierte Insulin eine gewisse Dauer-Aktivität behält und somit noch auf die Homöostase des Blutzuckerspiegels während der nächsten Mahlzeit Einfluß nehmen kann. Nach der Abendmahlzeit läßt die länger andauernde Steigerung des Insulingehaltes auf das Bestehen einer erneuten gleichzeitigen Insulinsynthese schließen.

Radioimmunoassay of estrone and estradiol-17β in peripheral plasma of pregnant and non-pregnant women

Abstract Estrone and estradiol-17β have been measured in non-pregnancy plasma, by radioimmunoassay, after Chromatographie separation on short Sephadex LH 20 columns. Dextran coated charcoal has been used for adsorbing unbound estrogen in the incubation mixture. No meaningful results could be obtained by measurement of total free estrogens in crude extracts of non-pregnancy plasma. In non-pregnancy plasma, the limited specificity of the antibodies imposed the chromatography step. The precision of the measurements during the menstrual cycle was of 9–15% (coefficient of variation). In pregnancy plasma the direct measurement of free estrogens in crude extracts, without chromatography, gave results that were proportional to the volume of plasma extracted. The precision of the measurements varied between 7–15% (coefficient of variation), for both low and high plasma concentration. In pregnant subjects a good correlation was found between the level of unconjugated estrogens in the plasma and the level of urinary estriol.

Accuracy of Endometrial Biopsy Dating in Relation to the Midcycle Luteinizing Hormone Peak

The accuracy of endometrial biopsy dating was evaluated in a selected group of apparently normal women in whom the basal body temperature (BBT), the onset of subsequent menstruation, the midcycle luteinizing hormone (LH) peak, and the 17β-estradiol peak were determined. Forty-two women with regular cycles, normal ovaries at laparoscopy, and luteal phases of 12 to 15 days were studied. In group I (n = 20), the infertility could be explained satisfactorily by either tubal occlusion or infertility of the husband, while in group II (n = 22) no explanation was found for the infertility. In group I, the duration of the luteal phase, defined as the interval between the LH peak and the onset of subsequent menstruation, was 13.4 ± 0.7 days. The plasma 17β-estradiol concentration declined (P It is suggested that, in women with a progressive rise in BBT over several days, the localization of the LH peak can be helpful for correct interpretation of the endometrial biopsy.

Slowly exchangeable pool of estradiol in the rat uterus

Abstract Chase experiments, alternating infusions with 14C and 3H labelled estradiol, were performed in adult rats. After infusion, purified nuclei were extracted from uterine homogenates and the 3H/14C ratio was determined in cytosol and nuclear fractions. It was shown that {414C}estradiol exchanged progressively with {6,73H}estradiol in all subcellular fractions, but at a much slower rate than in the plasma. The retardation was more evident in the Tris-EDTA-KCl extract (SIII) of the nuclear preparations than in other soluble “receptors” from the cyctosol (SI) or the particulate fraction (SII). The final residual precipitate of the nuclear fraction contained a limited pool of estradiol, more slowly exchangeable than in other fractions. These data confirm the extensive recycling of estradiol within the uterine cell. They also show in the nucleus, the existence of a more slowly exchangeable pool, of limited capacity, which appears to be possibly a final but still reversible step in the hormone distribution within the cell. The implication of this limited pool in the hormonal activity is briefly discussed.

“Unboun” ligand dsorption on dextran-coated charcoal: Practical consideration

Abstract Some practical aspects of estrogen binding to Dextran-coated charcoal were studied. The binding capacity of the charcoal was very large and not limitative in most instances. The affinity depended on its degree of dispersion in the medium. At 40 mg% of charcoal less than 2% of free [ 3 H]estradiol ( 3H -E 2 ) remained in the supernatant. At high concentrations (more than 200 mg%), charcoal was able to precipitate most of the BSA bound 3 H-E 2 , but none of the 3 H-E 2 bound to rat uterus cytosol prepared in Tris-EDTA buffer. BSA added to rat uterus cytosol decreased the amount of 3 H-E 2 not precipitated with charcoal. Charcoal was able to remove 3 H-E 2 or 3 H-E 2 from some specific antibodies, but not from others. Time and temperature of incubation in the presence of charcoal were critical in this respect. Precipitated charcoal was still able to adsorb the estrogen moiety, slowly released from the antibody in function of time. Practical considerations are discussed concerning the use of dextran-coated charcoal in radioimmunoassay procedures.

Subcellular distribution and binding of {6,7-3H} estradiol in rat uterus, at equilibrium, after long-term intravenous infusion

Abstract In the course of a 3 h-infusion of adult rats with {6,7- 3 H} estradiol, the radioactivity in the uterus was rapidly distributed between the cytosol and the particulate fraction. More than 50% was associated with the latter after 5 min of infusion; this proportion increased to a constant value of about 75% after 3 h. On sucrose density gradient containing 0.4 M KCl, the radioactivity in the cytosol was found associated with a 4–5 S “receptor”. On KCl free gradient, the bound radioactivity appeared in the form of aggregates and of a 4–5 S peak. From the particulate fraction, two distinct constituents were obtained: a 3.5–4 S binder, extracted with Tris-EDTA, pH 8.2, and a 4–5 S binder extracted thereafter with Tris-EDTA-KCl pH 8.5. The 3.5–4 S, Tris EDTA extractable “receptor” was more labile than the 4–5 S cytosol or the 4–5 S nuclear “receptor”. Its physiological significance is not yet clear. A significant amount of radioactivity remained associated with the residual precipitate. The data show that estradiol is rapidly distributed in the subcellular fractions and a constant partitioning between cytosol and particulate fraction is established, whatever the level of uterine concentration of the hormone. This suggests the existence of a dynamic exchange between the binding constituents at the subcellular level.

Dynamic exchange of {6, 73H}estradiol-17β at the subcellular level in rat uterus

Abstract In a previous work it was shown that {6, 7 3 H}estradiol, administered as a continuous intravenous infusion to adult rats, was taken up by the uterus and partitioned rapidly between cytosol and paniculate fractions, where it was bound to specific “receptors”. The rapid passage to the particulate fraction was followed by a slower equilibration leading to a constant ratio of radioactivity of about 25 and 75 % in cytosol and particulate fractions respectively. The dynamic equilibrium which was suggested from these data was funher analyzed by chase-experiments. Adult rats were first submitted to 3 h-intravenous infusions with radioinen estradiol, at a rate of 200 ng/h, in order to saturate the uterine binding sites; the infusion was then switched over to {6, 7 3 H} estradiol at the same rate, for 1–180 min. Uterine radioactivity increased progressively; however, contrary to plasma free radioactivity which had reached its equilibrium level after 45 min, the steady state was not achieved after 180 min. Subcellular partition of the labelled hormone in the uterus however was effected in a similar way as during radioactive infusions alone, without a preliminary saturation of the uterus with radioinen hormone. Binding to the “receptor” was manifest after only 2 min of {6,7 3 H}estradiol infusion. In vivo-in vitro chase experiments were performed by alternating 3 h-intravenous infusion with {6, 7 3 H}estradiol and 2–60 min incubation of the uteri with 5 × 10 −9 M radioinen estradiol, or vice versa. Estradiol bound to the uterus in vivo was slowly exchanged with the hormone from the medium. In course of the incubation the subcellular distribution of the former did not change. The subcellular distribution of the latter showed a slightly higher proportion in the cytosol up to 20 min of incubation. These data show that estradiol taken up by the uterus remains exchangeable with the circulating hormone. Also, a continuous dynamic exchange of estradiol exists between eytosol and paniculate fractions, resulting in an intracellular recycling of the hormone. The paniculate fraction however shows some retardation in the complete equilibration with the cytosol suggesting the existence in the former of a more slowly exchangeable pool.

Plasma levels of unconjugated estrogens in normal and diabetic pregnancies

Abstract Plasma ether-extractable estrogens were determined by radioimmunoassay which, based upon utilization of an antibody to estradiol-17β-hemisuccinate-bovine serum albumin, measured predominantly estradiol. Then 287 plasma samples from 215 normal pregnancies and 93 samples from 14 pregnant diabetic women were analyzed. The plasma estrogen (estradiol) concentrations found in the 14 diabetic patients fell within the range of levels observed in normal pregnant women at comparable periods of gestation. Serial plasma estrogen levels measured in one diabetic patient did not fall concomitantly with a decrease in urinary estriol/creatinine ratios determined simultaneously, suggesting that further evaluation is required before plasma estrogen assays may be accepted for clinical management.

New combined oral contraceptive with incremental progestogen dosage regimen

n In a study of 506 women, 272 took a total of 2124 cycles of combined preparations of norgestrel and 234 women took a total of 2002 cycles of a combined contraceptive with incremental progestogen doses, in which the total D-norgestrel was 1800 g as opposed to 5250 g in the combined preparation. The step-up preparation group had a similar rate of side effects as the combined preparation group; however, women using the step-up preparation had significantly diminished intermenstrual bleeding after the 6th month of use. The step-up regimen has not induced any cases of endometrial hyperplasia but does have an inhibiting effect on endometrial proliferation in the 1st half of the cycle. The step-up preparation has similar effects on the hypothalamo-hypophysial-ovarian axis as the combined pill. The step-up preparation appears to inhibit ovulation from the 1st cycle as indicated by the absence of LH and FSH midcycle peaks and the low 17 beta-estradiol levels in the 2nd half of the cycle; therefore, the step-up preparation should be classified as a new combined, not sequential, oral contraceptive.n

The “Biological Exchange Constant” of Estradiol-17β between Plasma and Uterus in the Adult and Immature Rats

Publisher Summary In vivo studies have been directed to the mechanisms by which estradiol-17β is retained in the target tissue after injection in the living animal. Little is known about the mechanism of uptake and of exchange with the circulating hormone. This requires a steady state situation approximating the physiological state. The present study aims to find whether a correlation existed between tissue levels of estradiol-17β, 6,73H and plasma levels of the hormone according to the simplified model. Free estradiol in the plasma exists as bound to nonspecific sites to specific sites. A small amount is unbound. In the tissue, similar compartments exist. Conjugates are abundant in the plasma and negligible in the uterus. Equilibrium between all these physical forms of the hormone is dependent on the relative values of the association constants. Considering the system as resulting from an equilibrium between bound hormone, and unbound hormone, the bound versus unbound level may be studied, according to the law of mass action, applied in the simplest situation corresponding to the interaction between one set of sites and one ligand.