John R. K. Preedy

Long-Term Effect of a First Pregnancy on the Secretion of Prolactin

An early first pregnancy is known to protect against subsequent breast cancer. We speculated that this effect may be mediated by a long-term depression of prolactin secretion after pregnancy. We therefore measured basal and post-stimulation serum levels of prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in two groups--15 women 18 to 23 years of age and 9 women 29 to 40--before and after a first full-term pregnancy, and in 40 appropriate nulliparous controls. We observed no significant change in basal levels of serum LH or FSH or in the levels stimulated by gonadotropin-releasing hormone in any group. A significant decrease was seen, however, in basal and perphenazine-stimulated levels of prolactin after pregnancy in both the younger and older first-pregnancy groups but not in the controls. In a separate cross-sectional study, we compared basal serum prolactin levels in 29 parous and 19 nulliparous women of similar age. The serum prolactin levels were significantly lower in the parous group but were not related to the number of pregnancies (one to three) or the time elapsed (12 to 150 months) since the last delivery. We conclude that a first pregnancy leads to a long-term decrease in serum prolactin secretion, lasting at least 12 to 13 years.

Comparative specificity of antisera raised against estrone, estradiol-17β and estriol using 6-0-carboxymethyloxime bovine serum albumin derivatives

Abstract Antisera for the radioimmunoassay of estrone and estradiol-17β in plasma are usually raised against estradiol-17β coupled to a protein through a derivative at carbon 17. Such antisera cross react with other naturally occurring estrogens, necessitating preliminary chromatographic separation. This difficulty could be overcome by the use of more specific antisera. We have raised antisera against the 6-0-carboxymethyloxime-bovine serum albumin (BSA) derivatives of estrone, estradiol-17β and estriol respectively. We have determined cross reactions with a number of estrogens and other naturally occurring steroids, and have compared the cross reactivity with that of an antiserum raised against estradiol-17β-17-succinyl-BSA. The former antisera show greatly reduced cross reaction with naturally occurring estrogens known or thought to be in relatively high concentration in plasma, as compared with the latter antiserum, but at the expense of greatly increased cross reaction with estrogens substituted at carbon 6. However, since these latter estrogens are thought to be in low concentration in plasma, the use of antisera raised against the 6-0-carboxymethyloxime-BSA derivatives should result in a net gain in specificity. The antisera raised against the estrone and estriol 6-0-carboxymethyloxime-BSA derivatives should be particularly useful.

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.

Separation of estrogen conjugates by high pressure liquid chromatography

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.

Metabolism of arterial plasma estrogens by the splanchnic organs of the dog in vivo.

In order to study the splanchnic metabolism of blood-borne estrogens, a constant infusion of estrone-6,7-(3)H was made in a series of dogs, and arteriovenous (A-V) differences at equilibrium were determined for estrone-6,7-(3)H and for its products estradiol-17beta, estrone sulfate, estrone glucosiduronate, and estradiol-17beta glucosiduronate across the splanchnic bed (artery-hepatic vein), the small intestine (artery-superior mesenteric vein), and the spleen (artery-splenic vein). Per cent extractions (100 - [V/A] 100) were calculated. The plasma metabolic clearance rate (MCR) for estrone was measured. Principal findings were as follows: mean MCR was 731 liters/day per m(2), SEM 50. By comparison with estimated hepatic plasma flow and using the observed splanchnic extraction of estrone, 45-71% of estrone metabolism was calculated to be extrasplanchnic. The significant mean per cent extractions were as follows (SEM in parentheses): splanchnic bedestrone 85.9 (1.92), estradiol-17beta 88.11 (3.36), estrone sulfate 27.9 (5.22), estrone glucosiduronate -48.5 (9.33), estradiol-17beta glucosiduronate -33.3 (80.3); small intestine-estrone 45.3 (2.60), estradiol-17beta 46.1 (12.9), estrone glucosiduronate - 30.8 (7.9); spleen-estrone 35 (3.8), estrone glucosiduronate 12 (3.7). These results lead to the following conclusions. Both estrone and estradiol-17beta are nearly completely extracted in one passage through the splanchnic bed. There is net uptake of estrone sulfate and net production of estrone glucosiduronate and of estradiol-17beta glucosiduronate by the splanchnic bed. There is net uptake of estrone and of estradiol-17beta by the intestine, associated with substantial net production of estrone glucosiduronate. There is net uptake of estrone by the spleen and a small but significant net uptake of estrone glucosiduronate.

Urinary excretion of estrone glucosiduronate, 1713-estradiol-17-glucosiduronate, and estriol-16a-6lucosiduronate. significance of proportionate differences during the menstrual cycle

The urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate in men and throughout the menstrual cycle in women was measured by specific radioimmunoassay. In 9 men the mean +/- SE excretion of these conjugates was 15.9 +/- 1.4, 2.7 +/- 0.3, and 3.2 +/- 0.2 microgram/24 h respectively. In 15 women studied in the midfollicular phase (day 8) of the menstrual cycle, the excretion was 19.4 +/- 1.7, 2.9 +/- 0.2, and 5.4 +/- 1.3 micrograms/24 h. Excretion of each conjugate was significantly (P less than 0.01) elevated in the midluteal phase (day 22) to 41.9 +/- 3.9, 6.3 +/- 0.8, and 12.2 +/- 1.5 micrograms/24 h respectively (n = 14). The mean excretion of estriol-16 alpha-glucosiduronate was greater than that of 17 beta-estradiol-17-glucosiduronate in the luteal phase (P less than 0.05) but not in the follicular phase or in men (P greater than 0.05). The excretion of each of these specific conjugates measured throughout the menstrual cycle in 7 women was characterized by a sharp midcycle peak and a lower, broader luteal phase peak. The ratios of estriol-16 alpha-glucosiduronate to 17 beta-estradiol-17-glucosiduronate, estrone glucosiduronate to 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate to estrone glucosiduronate throughout the menstrual cycle were analyzed. When the mean ratio during the follicular phase was set at 1, a significant increase (P less than 0.01) occurred in the mean luteal phase ratio in each case: 1.00 +/- 0.03 to 1.66 +/- 0.09, 1.00 +/- 0.04 to 1.30 +/- 0.04, and 1.00 +/- 0.03 to 1.24 +/- 0.04 (mean +/- SE) respectively. The marked alteration in the proportions of these urinary estrogen conjugates may be due to altered metabolism of 17 beta-estradiol, but it more likely reflects a change in the pattern of estrogen secretion or production between the two phases of the menstrual cycle.

Isocratic separation of estrogen conjugates on deae-sephadex

Abstract Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model y = a + b 1 x where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.

Extraction of estrogens by the hind limb of the dog. Evidence for entry into the lymphatics.

Substantial extrasplanchnic metabolism of estrogens is known to occur in humans and dogs. As part of an investigation into the anatomic sites of such metabolism, the extraction of estrogens by the hind limb of the dog was studied during a constant infusion of [3H]estrone. Simultaneous femoral artery (A) and femoral vein (FV) plasma samples were obtained and analyzed for total radioactivity, unconjugated and conjugated radioactivity, for [3H]estrone and for its metabolites estradiol-17β, estrone sulfate and estrone glucosiduronate. The percent extraction across the hind limb was calculated [100(1-FV/A)]. The mean percent extraction ± SE of total, conjugated and unconjugated radioactivity was 31 ± 3.9, 27 ± 4.4 and 16 ± 3.7 respectively, indicating significant net uptake of these moieties by the hind limb (P<.01). Mean percent extractions ± SE for estrone and estradiol-17β were 40 ± 4.9 and 32 ± 2.7, indicating significant net uptake of these specific unconjugated estrogens by the hind limb (P<.01). The mean percent extraction of estrone glucosiduronate was 16 ± 3.1 indicating significant net uptake of this conjugate (P<.01). However, the mean percent extraction of estrone sulfate was negative (−12 ± 4.1) indicating net production of this conjugate by the hind limb (P<.01). Since the net uptake of total radioactivity cannot be explained on the basis of metabolism by the hind limb, the lymphatics were investigated as an alternate efferent pathway. In similar experiments the thoracic duct was cannulated, the estrogens in lymph were analyzed and compared with those in femoral artery plasma. Each estrogen measured in plasma appeared in lymph within 10 minutes following the start of the [3H]estrone infusion. The lymph/femoral artery concentration ratios reached a plateau at 70–100 minutes after the start of the infusion. The plateau concentrations were 20–70% of those in plasma. It is suggested that removal of estrogens in the lymph may account, in part at least, for the net uptake of total radioactivity across the hind limb calculated from the plasma data.

A direct radioimmunoassay for estriol-16α-glucosiduronate: Its use in the determination of plasma and urine levels and renal clearance of this conjugate in pregnancy

A direct radioimmunoassay which does not require hydrolysis or chromatography has been developed for estriol-16alpha-glucosiduronate in pregnancy plasma and urine. The antigen used in the development of this antiserum was prepared by joining the carboxylic acid group of estriol-16alpha-glucosiduronate covalently to the epsilon-amino group of the lysine residues in bovine serum albumin. Results indicate that the direct radioimmunoassay yields levels of estriol-16alpha-glucosiduronate in late pregnancy urine that are comparable to those obtained by mroe elaborate procedures involving chromatographic separation of the estriol conjugates, followed by enzymic hydrolysis and measurement of the freed estriol. In addition, good correlation was found between the total estrogen values in urine obtained by currently used chemical procedures and urinary levels of estriol-16alpha-glucosiduronate. The renal clearance of estriol-16alpha-glucosiduronate was determined in eight normal women during the third trimester, and the mean value +/- S.D. was 404 +/- 81 ml. per minute (range, 248 to 494). This method is suitable for the evaluation of variations in the plasma and urine levels of estriol-16alpha-glucosiduronate in pregnancy and offers significant advantages over the presently used chemical methods for monitoring fetal well-being.

Prolactin and Blood Pressure Responses to Perphenazine in Human Subjects: Comparison of the Oral and Intramuscular Routes

Although several phenothiazines are known to stimulate prolactin (PRL) secretion, only chlorpromazine is in general use for this purpose in humans. However, chlorpromazine has severe sedative and hypotensive effects. Therefore, the effects of perphenazine on human PRL release and on blood pressure were evaluated. Perphenazine was administered orally (8mg) and intramuscularly (5mg and 2mg) to determine the optimal route and dose for evaluating PRL release. The postural hypotensive effect of perphenazine was evaluated with the 2mg intramuscular (IM) dose. The mean time of peak PRL response (hr ± SD) was significantly shorter (p<0.05) for the 5mg IM (1.7 ± 0.4) than the oral (4.5 ± 0.6) rout. Also, the mean ratio of peak/baseline PRL was significantly greater for the 5mg IM (8.87 ± 5.69) than the oral (5.12 ± 2.90) route. The major side-effect produced by perphenazine was drowsiness, which was moderate to severe with the 5mg IM dose. A lower IM dose (2 mg) retained PRL releasing activity, reduced drowsiness, and did not produce hypotension. For clinical testing, intramuscular perphenazine is preferred over oral perphenazine because of the shorter latency period and the higher PRL levels. Intramuscular perphenazine (2mg) is preferred to chlorpromazine since it did not produce a clinically significant hypotensive effect. This is the first report on the dynamic responses of PRL and blood pressure to intramuscular perphenazine in humans.