β-LIPOTROPHIN AND β-ENDORPHIN PLASMA LEVELS DURING PREGNANCY

β-lipotrophin (β-LPH) and β-endorphin (β-EP) plasma levels were measured by radioimmunoassay after glass powder extraction and Sephadex G-75 column chromatography in plasma samples from controls (ten healthy males and twenty-six young women in early follicular phase), from eighty-two pregnant women in weeks 9–40 after their last menstrual period, from nine women just after delivery and the cord blood of their neonates, in fifteen mixed cord blood samples and in seven amniotic fluid samples obtained by amniocentesis. No sex differences were found in β-LPH (120·6 ± 8·5 pg/ml) or β-EP (31·1 ± 2·4 pg/ml) plasma levels or in their molar ratio (1·34 ± 0·09) (MR). β-LPH plasma levels increased in early pregnancy (13–16 weeks) (185·0 ± 27·1 pg/ml) and remained high until weeks 21–24, then declining to levels similar to those of controls. β-EP plasma levels were significantly depressed in weeks 9–12 (20·7 ± 5·3 pg/ml), subsequently increasing to a maximum at weeks 36–37 (42·7 ± 6·8 pg/ml). β-LPH/β-EP molar ratio was about double normal in early pregnancy and decreased to normal in the second half. The present data indicate that β-LPH and β-EP present different patterns throughout pregnancy and that β-EP levels increase progressively, reaching the highest concentrations at term. At delivery, both β-LPH and β-EP showed maximum values (β-LPH: 230·2 ± 20·4 pg/ml; β-EP: 78·0 ± 7·4 pg/ml) and a MR of 1·02 ± 0·10 indicating that stressful situations, such as labour, stimulate a simultaneous rise in β-LPH and β-EP plasma levels. Cord blood specimens showed a wide range of values (β-LPH:75–347 pg/ml; β-EP: 16–287 pg/ml) with a MR of 1·21 ± 0·14. Amniotic fluid samples obtained late in the third trimester of pregnancy were characterized by β-LPH levels of 119·4 ± 26·4 pg/ml and β-EP levels of 29·6 ± 7·5 pg/ml.     

CORRELATIONS BETWEEN PROLACTIN AND PROGESTERONE, OESTRADIOL-1 7-β AND OESTRIOL DURING EARLY HUMAN PREGNANCY

The correlations between serum prolactin and progesterone, oestradiol-17-β and oestriol were determineed in 125 pregnant females between the sixth and fourteenth week of pregnancy. No significant correlations were found between prolactin and progesterone. Correlations between prolactin and oestradiol-17-β and oestriol were mostly positive, but only significant during the twelfth week of pregnancy.     

INCREASED PLASMA CONCENTRATION OF N-TERMINAL β-LIPOTROPHIN AND UNBOUND CORTISOL DURING PREGNANCY

The plasma concentration of N-terminal β-lipotrophin (β-LPH), total and protein unbound cortisol, progesterone and the transcortin (CBG) binding parameters have been measured in 21 women in the early follicular phase and in 70 pregnant women at various stages of pregnancy. Results showed that the plasma CBG binding capacity and the concentrations of total cortisol and progesterone increased significantly at each trimester of pregnancy while the plasma concentration of unbound cortisol increased significantly only in the 2nd and the 3rd trimesters of pregnancy. In addition, a significant increase of N-terminal β-LPH level was observed during the 3rd trimester. By chromatography, it is demonstrated that during the 3rd trimester of pregnancy the β-LPH/γ-LPH molar ratio decreases dramatically and that the increase of N-terminal β-LPH concentration is mainly due to a two fold increase in γ-LPH concentration.     

THE RELATIONSHIP BETWEEN 17β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY AND OESTROGEN CONCENTRATIONS IN HUMAN BREAST TUMOURS AND IN NORMAL BREAST TISSUE

The activity of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) was measured in human breast tumours and in normal breast tissue from premenopausal, perimenopausal and postmenopausal women. Enzyme activity was higher in tumour tissue than in normal tissue from the same breast and under the conditions of the assay the oxidation of oestradiol was higher than the reduction of oestrone. The physiological status of the women in the study did not relate to the activity of the enzyme in either normal or tumour tissue although fibroadenomas had less activity than adenocarcinomas. In postmenopausal women tumour tissue oestrogens were 2-3 fold higher than in normal tissue from the same breast. Furthermore, tumour tissue concentrations of oestradiol tended to be higher than those of oestrone although in normal tissue the two oestrogens were present in similar concentrations. In plasma from the same women oestrone was the predominant oestrogen. There appears to be no direct relationship between 17 beta HSD activity and oestrogen concentrations but the enzyme may play a part in determining the balance between oestrone and oestradiol according to substrate and cofactor availability.     

β-ENDORPHIN AND β-MSH IN HUMAN PLASMA

The aim of this study was to establish whether or not a peptide with chromatographic and immunological properties of beta-endorphin exists in human plasma. Using direct chromatography under conditions designed to minimize generation of beta-endorphin and beta-MSH from beta-LPH, we invariably found a peptide with beta-endorphin immunoreactivity eluting in the position of beta h-endorphin on gel chromatography in samples of plasma from patients with elevated ACTH and LPH levels. beta-MSH was only found in the plasma of one patient with the ectopic ACTH syndrome.     

PLASMA UNCONJUGATED OESTRIOL AND UNCONJUGATED OESTRADIOL-17β IN LATE PREGNANCY: INDIVIDUAL FLUCTUATION OF CONCENTRATIONS FROM DAY TO DAY AND EVERY 10 MINUTES

Plasma unconjugated oestriol (E3) and unconjugated oestradiol-17 beta (E2) were determined by radioimmunoassay. In ten normal women in their last month of pregnancy the individual fluctuation of E3 concentrations (mean of the coefficients of variation, CV) from day to day (over 5 days) was 15.6% (range 6.4--26.2%). The individual fluctuation of E2 determined in eight of these women was 16.9 (10.4--25.5) %. In six of the same women who had blood samples collected every 10 min (for 3 h) the individual fluctuation of E3 concentrations was 13.8 (7.0--25.1) %, and of E2 was 16.1 (11.3--26.0) %. The degree of fluctuation of E3 in individuals was in proportion to the mean concentration, unlike E2, suggesting that in clinical practice individual changes in E3 values would be as easy to interpret at low levels as at high levels. The finding that concentrations of plasma unconjugated E3 fluctuate in individuals no more than for E2 or than reported for total E3, and less than reported for 24-h urinary oestrogens, lends practical support to the theoretical preference for the assay of plasma unconjugated E3 to assess fetoplacental function.     

SUBCELLULAR PROGESTERONE IN HUMAN MYOMETRIUM DURING LATE PREGNANCY

Samples of myometrium were obtained from twenty-two patients undergoing caesarian sections for various obstetric indications. The myometrium was homogenized and divided into four subcellular fractions (crude nuclear, mitochondrial, microsomal and cytosol). The concentration of endogenous progesterone in these fractions was determined by radioimmunoassay. In addition the concentration of oestrone and oestradiol of fifteen cytosolic fractions was determined. The subcellular concentration of progesterone (pg/mg protein) in human myometrium was higher in samples taken during labour than in samples taken at elective caesarian sections. This finding was statistically significant in the crude nuclear (P < 0.005) and the microsomal (P < 0.05) fractions. The absolute concentration of progesterone was lowest in the nuclear and highest in the microsomal fraction. The relative progesterone concentrations in the four subcellular fractions were the same in both groups. The data show that there is no significant decrease in myometrial progesterone associated with labour in man. The mean concentration of oestrone was higher than the mean oestradiol concentration in the cytosol fraction of human myometrium in late pregnancy. This study shows that a completely different oestrogen ratio exists in myometrium than in plasma.     

MEASUREMENT OF PROGESTERONE IN HUMAN PLASMA

A rapid protein-binding technique is described for routine plasma progesterone measurements. Chick plasma is the source of binding-protein and ³H-progesterone is the radioligand. Accuracy is ensured by including a recovery determination with each specimen. In fifteen normal female volunteers, low plasma progesterone values-0.65 ng/ml ± 0.82 (SD)-were recorded during the follicular phase of the menstrual cycles, rising 10-14 days before menstruation to peak levels of 9–17 ng/ml during the luteal phase and then falling abruptly towards the end of the cycle.     

EXCRETION OF 6-OXYGENATED METABOLITES OF PROGESTERONE AND PREGNANEDIOL DURING PREGNANCY

The excretion of preganediola and the 6-oxygenated metabolites of progesterone was measured during the last 12 weeks of pregnancy in fifty-five women. There was no significant change in pregnanediol excretion during the last 10 weeks of pregnancy but during this time there was a significant increase in the excretion of the 6-oxygenated metabolites. There was a significant correlation between the excretion of these latter metabolites and both fetal and placental weights at term. It is suggested that the 6-oxygenated metabolites may be arising from the fetal 6-hydroxylation of progesterone. In some patients with severe toxaemia pregnanediol excretion was low although excretion of 6-oxygenated metabolites was within the normal range.     

A RAPID 5 HOUR RADIOIMMUNOASSAY OF PROGESTERONE AND OESTRADIOL IN HUMAN PLASMA

A rapid 5 h radioimmunoassay method for the determination of progesterone and oestradiol in the plasma of non-pregnant women is described. Due to the high specificity of the antisera used, it is possible to perform the radioimmunoassay directly on the ether extracts of plasma, without employing chromatographic purification of the steroids. Evidence is presented indicating that the rapid assay is almost as reliable as the previously described radioimmunoassay method which involves chromatography. The within-assay and between-assay coefficients of variation in the progesterone assay were 7-74 and 14-9 and in the oestradiol assay 7-36 and 18-1 respectively. Comparisons between increasing doses of authentic hormone and endogenous hormone extracted from plasma indicated no deviation from parallelism. Progesterone and oestradiol were assayed in 300 plasma samples by the rapid method and by the method involving chromatography. The slopes obtained by a regression analysis were close to unity for both progesterone and oestradiol (1-04 and 1-06, respectively), the y-intercepts were - 0-21 and 0-16 and the correlation coefficients 0-98 and 0-88, respectively. When the data obtained by both techniques in fourteen menstrual cycles were compared, the results were practically identical. In ten repeated studies conducted by four investigators it was shown that two workers can complete the assay of both progesterone and oestradiol in twenty-five plasma samples in duplicates with 5 h. The same time required for the assay of either progesterone or oestradiol in twenty-five duplicates by one worker.     

SOMATOSTATIN AND OXYTOCIN INFUSION INHIBITS THE RISE OF PLASMA β-ENDORPHIN, β-LIPOTROPHIN AND CORTISOL INDUCED BY INSULIN HYPOGLYCAEMIA

The present study investigated the possible effect of somatostatin and oxytocin on the basal and stress-induced rise of beta-endorphin (beta-END), beta-lipotrophin (beta-LPH) and cortisol in the human. For this purpose somatostatin (4.1 micrograms/min for 120 min or oxytocin (0.4 micrograms/min for 120 min) was infused into two different groups of seven healthy subjects; 30 min after the start of the infusion, placebo or insulin (0.1 IU/kg body weight, B.W.) was injected on two different days. In a third experimental step, an insulin tolerance test was performed during saline infusion to evaluate stress-related effects on the different hormonal secretions under basal conditions. Plasma levels of beta-END, beta-LPH and cortisol were measured by radioimmunoassay. Extraction and chromatographic procedures preceded the assay for beta-END and beta-LPH. Neither somatostatin nor oxytocin significantly modified basal plasma levels of beta-END, beta-LPH and cortisol. However these treatments blunted the rise of the three hormones seen at 45 and 60 min during insulin-induced hypoglycaemia (P less than 0.01). These results indicate that somatostatin and oxytocin may influence the beta-END, beta-LPH and cortisol increase induced by stress in humans, without affecting their basal secretion.     

CLINICAL EVALUATION OF A RADIOIMMUNOASSAY FOR β-MSH-RELATED PEPTIDES (LIPOTROPHINS) IN HUMAN PLASMA

A radioimmunoassay for β-MSH related peptides (‘β-MSH’) which measures βh- and γh-lipotrophin (LPH) and β-MSH, was used to assess LPH status and the stability of ‘β-MSH’ in blood and plasma.‘β-MSH’ concentrations correlated well with ACTH levels in paired snap-frozen plasma samples from normal subjects, from patients with pathologically elevated hormone levels (except those with chronic renal failure), and during acute stimulation and suppression tests (r= 0.913, P > 0.001). The mean ratio of ‘β-MSH’ and ACTH levels in most situations was approximately unity. In chronic renal failure,‘β-MSH’ concentrations were significantly higher than ACTH (mean ratio 4.1). Lesser degrees of dissociation of hormone levels were also found in patients with the ectopic ACTH syndrome (mean ratio 2.8). Basal ‘β-MSH’ levels were uniformly elevated in patients with Cushing's disease after bilateral adrenalectomy, Nelson's syndrome, ectopic ACTH syndrome, and Addison's disease and overlapped the normal range in patients with untreated Cushing's disease. There was complete separation of ‘β-MSH’ levels in ACTH-dependent and ACTH-independent (adrenal tumour) causes of Cushing's syndrome. Endogenous ‘β-MSH’ immunoactivity was more stable than ACTH in blood and plasma. There was no significant decrease of ‘β-MSH’ immunoactivity in samples kept at ambient temperature for 24 h or after four cycles of freezing and thawing plasma.‘β-MSH’ levels also did not change significantly in unhaemolysed blood samples transported at ambient temperature by first class mail. It is concluded that the ‘β-MSH’ assay provides at least as good discrimination as the ACTH assay in situations in which the latter is diagnostically valuable. Determination of the ratio of ‘β-MSH’ to ACTH levels may help to distinguish between untreated Cushing's disease and the ectopic ACTH syndrome. The stability of ‘β-MSH’ immunoactivity allows unhaemolysed blood and plasma samples to be transported at ambient temperature without loss of diagnostic value.     

HUMAN α-LACTALBUMIN AND HORMONAL FACTORS IN PREGNANCY AND LACTATION

Serum α-lactalbumin was monitored throughout pregnancy in twelve women and in a separate group of nineteen women during the first 3 months postpartum. During pregnancy α-lactalbumin rose significantly until the mid trimester (P < 0·001). From then until term, concentrations remained stable. Concentrations during labour were significantly higher (P < 0·01) than those seen at term, α-lactalbumin, 17β-oestradiol and progesterone concentrations behaved similarly during the first week of the puerperium in both lactating (n= 10) and non-lactating (n= 9) subjects. A large surge of α-lactalbumin closely followed the clearance of high circulating concentrations of sex steroids in both groups. Prolactin concentrations were significantly greater (P < 0·02) in lactating subjects by the third postpartum day. By the third postpartum week α-lactalbumin concentrations in lactating subjects had stabilized at labour levels in a milieu of high prolactin levels and depressed production on 17β-oestradiol and progesterone. Conversely, in non-lactating subjects α-lactalbumin concentrations fell, as did prolactin, coincidental with a rise in 17β-oestradiol, progesterone concentrations remaining barely detectable. The apparent control mechanisms for human α-lactalbumin secretion and thus, lactation, are discussed in the light of the data presented.     

PLASMA PREGNENOLONE, PROGESTERONE, 17-HYDROXYPROGESTERONE, TESTOSTERONE AND 5α-DIHYDROTESTOSTERONE IN DIFFERENT TYPES OF CONGENITAL ADRENAL HYPERPLASIA

Unconjugated pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5a-dihydrotestosterone were simultaneously measured by radioimmunoassay in plasma from children with congenital adrenal hyperplasia (CAH) due to a deficiency of 21-hydroxylase (21-OHase), 11 β-hydroxylase (11β-OHase) or 3β-hydroxysteroid dehydrogenase (3β-HSD). These steroids were also measured in a reference group of children of similar age. The following concentrations of these five steroids were observed in the prepubertal children aged 0-4-10-9 years: pregnenolone <0-2-0-85 ng/ml; progesterone 0-17-0-68 ng/ml; 17-hydroxyprogesterone 0-10-0-53 ng/ml; testosterone <0-05-0-14 ng/ml, and 5α-dihydrotestosterone <0-05 ng/ml (detection limit of the method). The concentrations were clearly elevated in the plasma of children with CAH. A very high plasma concentration of 17-hydroxyprogesterone differentiates a 21-OHase deficiency from the two other types: children with this defect had levels mostly in excess of 100-fold that of normal. Plasma progesterone concentrations in these patients were also high being in the range found during the luteal phase level in the adult. Their plasma testosterone concentrations showed a scatter from normal values to high adult male levels being mostly at the level of adult females. The concentrations of 5a-dihydrotestosterone were at or above those of adult males. A high plasma concentration of pregnenolone with at most slightly elevated progesterone and 17-hydroxyprogesterone levels differentiated the 3β-HSD defect from the other two forms of CAH. The plasma profile of the five steroids determined in a patient with an 11 β-OHase deficiency was close to normal, but slightly elevated pregnenolone, progesterone and 17-hydroxyprogesterone levels were found to be characteristic of this enzyme deficiency.     

INSULIN HYPOGLYCAEMIA AND CHOLINERGIC BLOCKADE: RESPONSE OF PLASMA IMMUNOREACTIVE β-ENDORPHIN

Six normal subjects were studied to investigate the mechanisms involved in the release of immunoreactive β-endorphin (ir-βEP) in response to insulin hypoglycaemia. Insulin alone (0·2 U/kg body weight) was followed after a 30 min lag period by a ? 2·5 fold elevation of plasma ir-βEP, with a return to basal levels by 90 min. Concurrent infusion of 10% dextrose at 250 ml/h for 2 h prevented the increase in plasma ir-βEP levels, suggesting an effect of hypoglycaemia rather than a direct effect of insulin. Premedication with atropine 0·6 mg at -30 min was followed by hypoglycaemia equivalent to that seen with insulin alone, but no increase in plasma ir-βEP, suggesting the involvement of cholinergic mechanisms in the neural mediation of ir-βEP release in response to hypoglycaemia.     

MEASUREMENT OF IMMUNOREACTIVE γ-MSH IN HUMAN PLASMA

A radioimmunoassay for immunoreactive γ-MSH (IR-γ-MSH) in human plasma has been developed. The assay is capable of detecting normal basal circulating levels which range from < 20-100 ng/1 at 0900 h. Plasma levels are raised concomitantly with ACTH during insulin induced hypoglycaemia and CRF stimulation and suppressed with dexamethasone. Chromatographic characterisation of IR-γ-MSH in plasma demonstrates a major peak of IR-γ-MSH, corresponding to purified glycosylated N-terminal pro-opiome-lanocortin 1-76, when IR-γ-MSH is secreted from the pituitary. In contrast IR-γ-MSH produced ectopically appears to be heterogeneous.     

THE RELATIONSHIP BETWEEN SALIVARY ALDOSTERONE AND PLASMA FREE ALDOSTERONE CONCENTRATIONS

The proportion of aldosterone in plasma not bound to proteins (% plasma free aldosterone, %PFA) was measured using an equilibrium dialysis technique and was found to lie predominantly in the range 25-40% although extremes of 20% and 60% were encountered. From this value and the total concentration of aldosterone in plasma (plasma aldosterone, PA) the concentration of free aldosterone in plasma (PFA) was calculated. Percentage PFA was not affected by the in-vitro addition of aldosterone (up to the equivalent of 2000 pmol/l) but the incremental addition of cortisol (up to the equivalent of 550 nmol/l) resulted in a linear increase in % PFA from 26% to 34%. Percentage PFA was found to be significantly, negatively correlated with serum albumin concentration (r = -0.87; P less than 0.001) but it was only when this was less than 15 milligrams that % PFA was abnormally high. Salivary aldosterone concentration (SA) was more highly correlated with PFA (r = 0.84) than with PA (r = 0.75), which indicates that even modest variance in %PFA has a discernible effect upon the overall SA-PA relationship. We conclude that measurement of salivary aldosterone concentrations gives a useful estimate of plasma free aldosterone level. Variations in the latter are determined more by changes in the total concentration of aldosterone in plasma than by variations in the degree of binding by plasma proteins.     

SERUM PREGNENOLONE, PROGESTERONE, 17α-HYDROXYPROGESTERONE, ANDROSTENEDIONE, TESTOSTERONE, 5α-DIHYDROTESTOSTERONE AND ANDROSTERONE DURING PUBERTY IN BOYS

We have determined the concentrations of pregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, 5α-dihydrotestosler-one and androsterone in serum samples collected from a total of seventy-nine boys who were 8–18 years of age at the time the first blood samples were drawn. Additional samples were drawn from sixty-six and forty-four of these boys after 1 and 2 year intervals, respectively. The first increases in serum steroid concentrations were those of androstenedione and androsterone, thus supporting the hypothesis that an early activation of the adrenal cortex is the first hormonal change in puberty. This increase in serum androstenedione occurred 2 years earlier than the first significant increase in serum testosterone, which took place by 13 5 years, after the first signs of external genital development, but in concert with the onset of pubic hair growth. The serum concentrations of 5α-dihydrotestosterone were closely related to those of testosterone, but the relative increases were smaller, the consequence of which is a decrease in the ratio of 5α-dihydrotestosterone: testosterone throughout puberty. The main increase in serum androsterone tends to take place after that of 5α-dihydrotestosterone and testosterone. In comparison with the four androgens, the serum concentrations of their C₂₁ precursors correlated rather poorly with the physical signs of advancing puberty. As in the case of androstenedione, the concentrations of pregnenolone and 17α-hydroxyprogesterone were relatively high in the youngest age groups, which probably also reflects an early adrenal activation. In contrast to testosterone, the major increases in precursor steroids occur at a relatively late stage of puberty. It seems likely therefore that a major qualitative shift in the testicular secretion of steroids occurs during puberty in boys.     

INCREASED PLASMA ACTIVITIES OF N-ACETYL-β-D-GLUCOSAMINIDASE ISOENZYMES IN HUMAN DIABETES MELLITUS

Plasma N-acetyl-beta-D-glucosaminidase activities are raised in maturity-onset insulin-dependent diabetic patients. Continuous gradient DEAE-cellulose column chromatography and assay of heat-stable enzyme activity both show that the activities of all isoenzymes are equally increased rather than a previously described increase of specific isoenzymes. Plasma enzyme activities are similarly increased in patients with and without diabetic retinopathy, and correlate with simultaneous plasma glucose concentrations.     

A COMPARISON OF THE EFFECTS OF CLOMIPHENE AND TAMOXIFEN TREATMENT ON THE CONCENTRATIONS OF OESTRADIOL AND PROGESTERONE IN THE PERIPHERAL PLASMA OF INFERTILE WOMEN

The effects of clomiphene and tamoxifen treatment on the concentrations of oestradiol and progesterone in plasma were compared in the same infertile women. Nine patients, three with anovulation and six with suspected luteal phase deficiency, were given clomiphene during 2 months and tamoxifen during 2 months. Placebo treatment was given in the month before the first drug and during a month between drug treatments. The concentrations of oestradiol and progesterone were determined by radioimmunoassay in three samples collected each month between days 6 and 8, 11 and 13, and 18 and 20. The mean concentration of oestradiol at the time of the expected pre-ovulatory rise was 0.82 nmol/l with placebo treatment, 1.20 nmol/l following tamoxifen treatment and 5.00 nmol/l after clomiphene treatment (normal menstrual cycle maximum, 2.0 nmol/l). The mean concentration of progesterone in the luteal phases reached maxima of 41 nmol/l, 47 nmol/l and >72 nmol/l, respectively (normal menstrual cycle maximum, 60 nmol/l). When the frequency distributions of hormone concentrations were examined for each treatment, clomiphene and tamoxifen were both found to alter the distribution from that of placebo treatment (Chi-square analysis), giving a larger proportion of high concentrations. In the six patients with suspected luteal phase deficiency clomiphene treatment was followed by biochemical evidence of ovarian hyperstimulation. There was no evidence of this when any of these patients were treated with tamoxifen, nor in anovulatory patients treated with clomiphene.