B. Little

Study of ketamine as an obstetric anesthetic agent

Abstract Ketamine [2-(o-chlorophenyl)-2-(methylamino) cyclohexanone monohydrochloride], a new short-acting anesthetic agent which does not inhibit laryngeal or pharyngeal reflexes, has been studied in 14 pregnant subjects and 18 nonpregnant controls. Approximate clearances calculated indicated a reduced clearance in pregnant subjects (48 cf. 64 ml. per minute per kilogram). Side effects included a 30 to 40 per cent increase in systolic and diastolic blood pressure, an increase in pulse and respiration, salivation, and nausea, and vivid but usually pleasant dreams. The fetal pH did fall after administration of anesthesia, but remained in the normal range along with Pco 2 and Pco 2 . Intrauterine tone increased, and there were changes in fetal heart rate, but none that might not have occurred with advancing labor. Newborn infants were not unduly depressed if the dose was kept below a priming dose of 1.5 mg. per kilogram followed by infusing 0.08 mg. per kilogram per minute. Serum bilirubin concentrations in the newborn infant were slightly increased and could not be accounted for. Mothers and babies all went home at the expected time post partum in apparent good health.

In vivo aspects of progesterone distribution and metabolism

The end-organ response of any hormone is the result of many factors which precede the event, including biosynthesis, secretion, transport, distribution, and metabolism. These factors vary among different species. The metabolic clearance rate (MCR) of progesterone varies between 40 and 180 L./day/Kg. in man (60 to 70), monkey (40 to 50), rabbit (55 to 60), sheep (110), rat (120), and guinea pig (180). Major sites of clearance include liver, brain, and uterus. Specific metabolites of progesterone include 20 alpha-hydroxypregn-4-en-3-one (20 alphaOHP) and alpha-pregnan-3,20-dione (5 alpha-DPH). Liver, brain, and uterine clearances, extractions, and conversions of progesterone to these metabolites have been studied in various species under apparent steady-state conditions. A specific hormone action of progesterone, the appearance of uteroglobin in the rabbit uterus, has also been studied in varying horomonal states (estrogen, estrogen plus progesterone, and progesterone alone). These have all been used as examples of progesterone distribution and metabolism.

In situ Estradiol and Progestin (R5020) Localization in the Vascularly Separated and Isolated Hypothalamus of the Rhesus Monkey

A neurosurgical procedure has been developed for vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. Utilizing this preparation, the left and right halves of the hypothalamus were perfused simultaneously, but separately, with a dextran-blood solution. Radiolabeled steroids were directly perfused in the dextran-blood into either the left or right half of the hypothalamus. Studies with radiolabeled gonadal steroids indicate that the majority of the carotid circulation is confined to the hypothalamus-thalamus area in this brain preparation and the cross-circulation of labelled steroids between the left and right sides of the hypothalamus is less than 10%. The usefulness of the preparation is illustrated by an autoradiographic study of the in situ hypothalamic distribution of (3H)estradiol in ovariectomized rhesus monkeys and of the synthetic progestin(3H)R5020 in estrogen-primed, ovariectomized rhesus monkeys. The direct perfusion of the (3H) steroids into the hypothalamus greatly increases the sensitivity of such compared to systemic administration of the (3H) steroids. The perfusion of one-half of the hypothalamus with )3H) steroid and the other half with (3H) steroids. The perfusion of one-half of the hypothalamus with (3H) steroid and the other half with (3H) steroids. The perfusion of one-half of the hypothalamus with (3H) steroid and the other half with (3H)steroid plus radioinert steroids permits in 1 animal, acting as its own control, the examination of a saturable distribution of a gonadal steroid in the rhesus monkey hypothalamus.

Autoradiographic Analysis of Progestin-Concentrating Cells in the Isolated Rhesus Monkey Hypothalamus

The in situ hypothalamic distribution of (3Η)progesterone was studied in the vascularly separated and isolated hypothalamus of estrogen-treated, ovariectomized rhesus monkeys. The distribution of (3H)progesterone in one half of the hypothalamus was compared with the distribution of the synthetic progestin (3H)R5020 in the contralateral half of the same hypothalamus. As an indication of the patency of the arterial circulation to, and throughout, the hypothalamus and to histologically identify the half of the hypothalamus perfused with (3H)progesterone, 15-μm radioinert microspheres were injected into one of the carotid arteries approximately 5 min before stopping the perfusion. The observed microsphere distribution confirmed the efficiency of the in situ perfusion system to deliver (3H)progestin throughout the preoptic area and hypothalamus. The hypothalamic distribution of (3H)progesterone and/or its metabolites was similar to that of (3H)R5020 although the cellular labeling by (3H)R5020 was more intense and more neurons within specific nuclei were labeled. The (3H)progestin-concentrating cells were localized along the midline of the medial preoptic area (sparse labeling), within the suprachiasmatic nucleus (sparse labeling), along the periventricular (PERI)-ventromedial nuclear borders in the mid-hypo thalamus, as well as in the ventromedial, infundibular (arcuate), premammillary and medial mammillary nuclei. Radio inert progesterone infused with the (3H)progesterone greatly diminished the intensity of labeling in all areas except for that localized in the PERI region. Radioinert cortisol infused with the (3H)progesterone also apparently decreased but did not eliminate the labeling. The results support the use of the synthetic progestin (3H)R5020 to evaluate the hypothalamic distri bution of progesterone in the rhesus monkey and also indicate a nonsaturable progestin labeling of the midhypothalamic periventricular-ventromedial border region.

Distribution of cholesterol side-chain cleavage and 11β-hydroxylase in the mitochondria of bovine adrenal cortex: Release by phospholipase A☆

Abstract The intramitochondrial distribution of steroid 11β-hydroxylation and cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria have been studied. Incubation of the mitochondria with phospholipase A followed by differential centrifugation yielded four fractions: supernatant (S3), a low-speed, compact pellet (R2), a low-speed, loosely packed pellet (“interphase”), and a high-speed pellet (K). Steroid 11β-hydroxylation and cholesterol side-chain cleavage activity could be recovered in good yield in the supernatant fraction and separated from the succinate dehydrogenase, succinate oxidase, cytochrome c oxidase and ATPase activities, and cytochrome (a + a3). By controlled digestion of the mitochondria, the cholesterol sidechain activity could be separated from the 11β-hydroxylation activity and these two activities were released into the supernatant fraction at different rates than were the adenylate kinase, malate dehydrogenase, and α-ketoglutarate dehydrogenase activities. It is concluded that the cholesterol side-chain cleavage activity is located in the outer membrane compartment of the bovine adrenocortical mitochondria. Electron microscopy supported this conclusion with regard to the distribution of the cholesterol side-chain cleavage system, but left unanswered the precise distribution of the steroid 11β-hydroxylase.

Autoradiographic localization of estradiol- and progesterone-concentrating neurons in the isolated rhesus monkey hypothalamus

Progesterone- and estradiol-concentrating neurons were autoradiographically localized in the in situ, vascularly isolated, rhesus monkey hypothalamus. Estradiol-concentrating neurons were dispersed throughout the hypothalamus, their density being greatest in the medial preoptic (MPOA) and medial basal hypothalamic nuclei (viz. dorsomedial, ventromedial (VMH) and infundibular nuclei). In contrast, progesterone-concentrating neurons were less densely localized in the medial preoptic, ventromedial (medial division) and infundibular nuclei. There was a virtual absence of progestin-concentrating cells in the anterior and posterior hypothalamic zones. This localization of cells was not attributed to the perfusion pattern of the isolated hypothalamus since a complete distribution of injected microspheres was found throughout the hypothalamus. The results of these studies indicate that a possible division between estradiol- and progesterone-concentrating neurons exists within the MPOA-medial basal hypothalamic nuclei of the rhesus monkey.

Isolation of placental and liver “mitochondrial” fractions which support steroid 11β-hydroxylation

Abstract 1. 1. Extracts of acetone powders of human placental or bovine liver “mitochondria” in the presence of bovine adrenal “mitochondrial” particles catalyze steroid 11β-hydroxylation 1 . The extracts have been subjected to (NH 4 ) 2 SO 4 fractionation, Sephadex G-100 gel filtration and DEAE-cellulose column chromatography, and fractions active in supporting 11β-hydroxylation were isolated and compared with adrenodoxin isolated from bovine adrenal “mitochondria”. 2. 2. The placental and liver DEAE-cellulose fractions did not have significant absorbence in the 400–700 nm region nor did they contain nonheme iron or acid-labile sulfur although their Chromatographic properties were similar to adrenodoxin.

In situ Subcellular Distribution and Metabolism of Progesterone, Estradiol and Androstenedione in the Vascularly Separated and Isolated Hypothalamus of the Female Rhesus Monkey

A neurosurgical procedure has been developed for the vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. The circulation to the left and right halves of the hypothalamus was also isolated and each half of the hypothalamus was perfused simultaneously, but separately, with a dextran-blood solution which contained radioactive gonadal steroids. The hypothalamus in situ efficiently converted [3H]androstenedione to [3H]estrone and this aromatization was inhibited by the presence of androsta-1,4,6-triene-3,17-dione (ATD) in the perfusate. [3H]Progesterone was metabolized predominantly to 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OHP). Subcellular fractionation of the hypothalamus after the in situ perfusion with [3H]-progestin or [3H]estradiol to the hypothalamus of estrogen-treated ovariectomized monkeys or oil-treated ovariectomized monkeys, respectively, indicated that the retention of [3H]estradiol in the nucleus was a saturable, limited-capacity phenomenon. No saturable subcellular distribution of [3H]progesterone or [3H]R 5020 was observed. This latter observation might be attributable to the presence of a progesterone receptor in too small a concentration to be detected by the methods used.

The head extractions, brain and pituitary uptake and metabolism of progesterone and 5α-dihydroprogesterone in anesthetized, female rabbits

Three days after ovariectomy adult female rabbits were injected intramuscularly with either 2 mug estradiol/kg b.w., 0.5 mg progesterone/kg b.w., 2 mug estradiol plus 0.5 mg progesterone/kg b.w., or oil vehicle daily for 5 days. On the sixth day the animals were anesthetized and given a continuous infusion of [14C]progesterone and [3H]5alpha-pregnane-3,20-dione (5alpha-DHP) for 4 h via the femoral vein. Head extractions, calculated from the difference in blood concentrations of radioactive steriods between the femoral artery and jugular vein, were in the range of 60-75% for both progesterone and 5alpha-DHP and were unaffected by the hormone treatments. Nine brain areas and the pituitary were analyzed for [14C]progesterone, [3H]5alpha-DHP and [14C]-5alpha-DHP. Generally, the brain and pituitary retention and distribution of [14C]progesterone and [3H]5alpha-DHP and the brain and pituitary metabolism of [14C]progesterone were unaffected by hormone treatments. In the cerebellum, progesterone treatment increased [14C]progesterone retention as compared to oil treatment. All tissues contained 2-7 times more [14C]progesterone than arterial blood concentrations. [3H]5alpha-DHP tissue: arterial blood concentrations ratios were much lower ranging from 0.9 to 1.78. [14C]progesterone and [3H]5alpha-DHP showed similar brain distribution with highest concentrations in the pons, pons reticulum and midbrain reticulum. All tissues converted progesterone to 5alpha-DHP, but only the cerebellum contained more [14C]5alpha-DHP than [14C]progesterone.

Progesterone and 5α-reduced metabolites: Facilitation of lordosis behavior and brain uptake in female hamsters

Ovariectomized, estrogen-primed female hamsters were tested for receptivity following injection of progesterone, 5 α -pregnan-3,20-dione (5-DHP), or 3 α -hydroxy-5 α -pregnan-20-one (3-5-P) in doses of 0.5, 1.2, or 2.4 mg. Progesterone facilitated lordosis behavior at all dosages. 5-DHP facilitated receptivity only at the highest dosage, and 3-5-P was totally ineffective in stimulating receptivity. In a second experiment, ovariectomized, estrogen-primed female hamsters received injections of either [ 3 H] progesterone or [ 3 H] 5-DHP. The accumulation of radioactivity in brain samples following [ 3 H] 5-DHP administration was only 17–45% of that found after[ 3 H]progesterone injection. However, chromatographic analysis revealed that [ 3 H]5-DHP levels were 64–105% of [ 3 H] progesterone brain levels. It was concluded that 5 α -reduction is not an important metabolic step in progesterone facilitation of receptivity in hamsters.