Pituitary Venous Effluent Research Articles

Maximizing the accuracy of Inferior petrosal sinus sampling: validation of the use of Prolactin as a marker of pituitary venous effluent in the diagnosis of Cushing’s disease

This study aimed to increase the accuracy of the inferior petrosal sinus sampling (IPSS) procedure and reduce the false-negative rate through the addition of prolactin as a marker of pituitary venous outflow as well as validate this adjunct to the test process. Inferior petrosal sinus sampling (IPSS) for ACTH is the current gold standard test for the differentiation of pituitary Cushing's disease from the ectopic ACTH syndrome. Although early studies with IPSS reported a diagnostic sensitivity and specificity approaching 100%, additional experience has revealed a false-negative rate of 1-10%. This has been attributed to either technical problems with unsuccessful petrosal sinus catheterization or anomalous venous drainage of the pituitary. Previous studies have suggested that the measurement of other anterior pituitary hormones may be useful during IPSS as a guide to the effectiveness of cannulation and to improve the diagnostic accuracy of the procedure. We reviewed the data, in this retrospective cohort study, for all patients who had undergone IPSS for the investigation of ACTH-dependent hypercortisolism. The study included 83 patients who underwent IPSS at St. Thomas's hospital between 2005 and 2010. Plasma ACTH and prolactin levels were measured both centrally and peripherally. The normalized ACTH/Prolactin inferior petrosal sinus/peripheral ratio was then calculated to assess the accuracy of the sampling procedure. A total of 83 patients with confirmed ACTH-dependent cortisol excess underwent investigation with IPSS during the study period. Sixty-seven patients initially had a positive IPSS result (i.e. a basal central/peripheral ACTH ratio >2·0 and >3·0 post-CRH). However, when the concurrent prolactin data were used, six patients were additionally found to have positive results suggestive of pituitary Cushing's. The Prolactin normalized ACTH IPS/Peripheral ratios were all >0·8 in patients with proven Cushing's disease, whereas they were all <0·6 in proven ectopic ACTH syndrome. The diagnosis was subsequently confirmed histologically in 72 of the patients. Using Prolactin as a concurrent index of pituitary venous effluent helps us recognize whether pituitary venous blood has been accurately sampled. Normalizing the IPS/peripheral ratios with Prolactin helps to improve the accuracy of the result and reduces the false-negative rate. With regards to the usefulness/validity of this test in clinical practice, it is relevant, reproducible and is easily adaptable from the existing diagnostic sequence.

Secretion of luteinizing hormone into pituitary venous effluent of the follicular and luteal phase mare: novel acceleration of episodic release during constant infusion of gonadotropin-releasing hormone

We tested the hypothesis that continuous infusion of native GnRH into mares during the estrous cycle, at a dose of 100 μg/h, would elevate circulating concentrations of LH without disrupting the endogenous, episodic pattern of LH release. Ten cyclic mares were assigned to one of two groups (n = 5/group): (1) Control (saline) and (2) GnRH in saline (100 μg/h). On experimental day 0 (3 to 6 d after ovulation), osmotic pumps containing saline or GnRH were placed subcutaneously and connected to a jugular infusion catheter. Blood samples were collected from jugular catheters daily and at 5-min intervals from catheters placed in the intercavernous sinus (ICS) for 8 h on experimental day 4 (luteal phase; 7 to 10 d after ovulation), followed by an additional 6-h intensive sampling period 36 h after PGF2α-induced luteal regression (experimental day 6; follicular phase). Treatment with GnRH increased (P < 0.001) concentrations of LH by 3- to 4-fold in the peripheral circulation and 4- to 5-fold in the ICS. Continuous GnRH treatment accelerated (P < 0.01) the frequency of LH release and decreased the interepisodic interval during both luteal and follicular phases. Treatment with GnRH during the luteal phase eliminated the low-frequency, long-duration pattern of episodic LH release and converted it to a high-frequency, short-duration pattern reminiscent of the follicular phase. These observations appear to be unique to the horse. Further studies that exploit this experimental model are likely to reveal novel mechanisms regulating the control of gonadotrope function in this species.

Failure of an Equine Homologue of Avian Gonadotropin Inhibiting Hormone to Alter Secretion of Luteinizing Hormone in the Mare.

The RF-amide family of peptides has been shown to regulate various physiological functions, including food intake and reproduction. In avian species, a RF-amide peptide with suppressive effects on the release of LH has been termed gonadotropin-inhibiting hormone (GnIH) and has been proposed to function as a major regulator of reproductive seasonality. In some mammalian species, GnIH and its mammalian homologue (RF-amide-related peptide; RFRP) have been shown to suppress the secretion of LH and adenohypophyseal responsiveness to GnRH. This has led to speculation of a role for RFRP in regulating seasonal-breeding in the horse. The sequence of the equine RFRP cDNA and analysis of predicted cutting sites for the equine prepro-RFRP indicate that 3 RF-amide-related peptides can be produced, with RFRP-3 as the homologue for the avian GnIH. Three experiments were conducted to test the hypothesis that eRFRP3 negatively regulates the secretion of LH in mares. Equine RFRP-3 (IPNLPQRF-G; MW 983) was synthesized (Auspep Pty Ltd, Tullamarine Victoria, AU) and used in all experiments. In Exp. 1, Alzet osmotic pumps delivering gonadotropin-releasing hormone (GnRH) continuously at a rate of 20 µg/h were placed s.c. in 6 mares 2 d after ovulation. Mares received bolus I.V. injections of 0, 500 and 1,000 µg eRFRP3 in a replicated Latin square design during Days 4, 6 and 8 post-ovulation. Jugular blood samples were collected before and at 15-min intervals for 4 h following treatment for assay of LH. For Exp. 2, the intercavernous sinus (ICS) of follicular phase mares was catheterized via the external facial vein for sampling of pituitary venous effluent and characterization of episodic LH release. Blood samples were collected at 5-min intervals for 6 h. Mares (n = 6 per treatment) received either saline or eRFRP3 (250 µg) I.V. every 10 min for 6 h beginning 2 h after onset of sampling. At hour 6, each mare was challenged with 1000 µg GnRH and blood sampling continued at 15-min intervals for the remaining 2 h of treatment. In Exp. 3, 3 winter anovulatory mares treated continuously with GnRH (100 µg/h), which elevated circulating LH to values similar to the breeding season, were used. On Day 5, the ICS was catheterized as in Exp. 2 and mares received saline or RFRP-3 (5 mg). ICS blood samples were collected at 5-min intervals for 3 h, with a 24-h washout period between treatments. In Exp. 1, mean concentrations (ng/ml) of LH in the peripheral circulation averaged 1.2 ± 0.2 ng/ml and did not differ between groups before or following RFRP-3 treatment. In Exp. 2, neither mean ICS concentrations of LH (1.3 ± 0.2 ng/ml), nor frequency (3.6 ± 0.55 episodes/h), amplitude (0.2 ± 0.03 ng/ml), and duration (36.3 ± 3.5 min) of individual secretory episodes differed between groups before or after eRFRP-3 treatment. Areas under the GnRH-induced LH curve (arbitrary units) also did not differ between control and treated mares (227.4 ± 42.9 vs. 260.2 ± 36.2). Finally, a single I.V. injection of 5 mg RFRP-3 (Exp. 3) failed to alter any variable of LH release. In contrast to observations in birds and other mammals, results of the current experiments fail to provide evidence for functional activity of eRFRP-3 in regulating LH release in the mare. Supported by Texas H-9137 and the Link Equine Research Fund. (poster)

Identification of Patients with Cushing’s Disease with Negative Pituitary Adrenocorticotropin Gradients during Inferior Petrosal Sinus Sampling: Prolactin as an Index of Pituitary Venous Effluent

Inferior petrosal sinus sampling for ACTH differentiates pituitary ACTH-dependent Cushing's (CD) from the ectopic ACTH syndrome (EAS). Petrosal sinus to peripheral (IPS:P) ACTH ratios greater than 2.0 in the basal state or a peak greater than 3.0 after CRH are diagnostic of CD. However, false-negative rates of 1-10% have been reported. We report three patients with features of CD with peak IPS:P ACTH ratios less than 3.0 after CRH suggesting EAS. We compared IPS:P prolactin (PRL) as an index of pituitary venous effluent in these three index cases with 44 patients with CD and five with EAS. The dominant basal IPS:P PRL ratio was greater than 1.8 in all 49 patients but was less than 1.2 in the three index cases. The IPS:P ACTH ratio normalized to IPS:P PRL was greater than 0.8 in all CD patients but was less than 0.6 in EAS patients. The IPS:P ACTH ratios normalized to IPS:P PRL were greater than 1.2 in the index cases, which was similar to those with CD. The three index cases had clinical and biochemical remissions after pituitary surgery.PRL is an index of pituitary venous effluent during inferior petrosal sinus sampling in patients with CD who fail to have a peak IPS:P ACTH ratio greater than 3.0 after CRH. IPS:P PRL should be measured when results indicate EAS.

The effect of endotoxin administration on the secretory dynamics of oxytocin in follicular phase mares: relationship to stress axis hormones.

The primary aim of this study was to define the secretory dynamics of oxytocin and vasopressin in pituitary venous effluent from ambulatory horses during acute endotoxaemia, a stimulus that may release both hormones. Our secondary aim was to investigate the role of oxytocin in regulating adrenocorticotropic hormone (ACTH) secretion by comparing oxytocin, vasopressin, corticotropin-releasing hormone (CRH) and ACTH secretory profiles during endotoxaemia and by monitoring the ACTH response to oxytocin administration. Pituitary venous blood was collected nonsurgically continuously and divided into 1-min segments from eight follicular phase mares. Four mares were sampled for 30 min before and 3.5 h after receiving an i.v. infusion of bacterial endotoxin (TOX). Four control mares were sampled for 2.5 h without infusion of TOX. Another three follicular phase mares were given 5 U of oxytocin to replicate the peak response to TOX and pituitary blood collected every 1 min for 10 min before and 15 min after injection. Endotoxin raised the secretion rates of all hormones measured. All hormones were released episodically throughout the experiment, with TOX increasing the amplitude of peaks in each hormone. Peaks in oxytocin and vasopressin were coincident in each treated mare. Similarly, ACTH peaks were coincident with peaks of oxytocin and vasopressin in each treated mare, and with peaks of CRH in three mares. However, oxytocin administration did not affect ACTH secretion. We conclude that during endotoxaemia in horses: (i) oxytocin and vasopressin are secreted synchronously; (ii) oxytocin is unlikely to be acting as an ACTH secretagogue since inducing peak oxytocin concentrations observed during TOX does not raise ACTH; and therefore (iii) the close relationship between oxytocin and ACTH secretion is circumstantial and due to the fact that oxytocin secretion is concurrent with that of vasopressin, a proven ACTH secretagogue in horses.

Concentrations of oxytocin in the intercavernous sinus of mares during luteolysis: temporal relationship with concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha.

The reproductive tracts of nine thoroughbred mares were examined by ultrasound to determine the day of ovulation (day 0). Mares were fitted with intercavernous sinus cannulae on the day before the start of sample collection of pituitary venous effluent rich in oxytocin. Intercavernous sinus blood samples were collected for at least 36 h at 5 min intervals beginning at noon on day 13 (n = 2), day 15 (n = 5) or day 16 (n = 2) after ovulation. Concentrations of oxytocin and 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) in plasma were determined by radioimmunoassay. Three high-magnitude surges of PGFM (> 1 ng ml-1) were found in these samples. Three high magnitude pulses of oxytocin (> 200 pg ml-1) were also observed, one associated with each of the PGFM surges. In each of these cases, the oxytocin pulse appeared to follow or coincide with the onset of the PGFM surge. Lower magnitude pulses of both hormones were detected throughout the bleeding period in every mare. The average interval between these pulses was 122.3 min for oxytocin and 121.0 min for PGFM. The interval between pulses for individual mares varied from 90 to 199 min for oxytocin, and from 87 to 213 min for PGFM. However, there was no correlation between PGFM and oxytocin pulse intervals among mares. Within each mare, there was no discernable association between low magnitude pulses of oxytocin and PGFM. From these data, it was concluded that high-magnitude surges of PGF2 alpha are associated with similar surges of oxytocin from the posterior pituitary gland, and that PGF2 alpha may induce their secretion. The posterior pituitary gland also appears to secrete oxytocin in a pulsatile manner at a frequency of approximately 1 pulse every 2 h but these pulses do not appear to be associated with the low magnitude pulses of PGF2 alpha secreted from the uterus.

A technique for collection of pituitary venous effluent for measuring pulsatile secretion of equine gonadotrophic hormones

A technique for collection of pituitary venous effluent for measuring pulsatile secretion of equine gonadotrophic hormones

The effects of cortisol, vasopressin (AVP), and corticotropin-releasing factor administration on pulsatile adrenocorticotropin, alpha-melanocyte-stimulating hormone, and AVP secretion in the pituitary venous effluent of the horse.

Plasma ACTH, arginine vasopressin (AVP), and alpha MSH were measured in pituitary venous effluent at 5-min intervals from five unanesthetized horses during cortisol infusion and after an iv bolus of AVP or ovine (o) CRF. In control experiments (no hormone) there was a significant overall correlation between the timing of concentration changes in ACTH and alpha MSH. Cortisol infusion increased jugular cortisol levels by 70% and was associated with a reduction in mean ACTH, AVP, and alpha MSH secretion rates and ACTH peak secretion rate, but did not alter the observed pulse frequencies of these hormones. Administration of AVP raised plasma concentrations to a level comparable to the spontaneous peaks in pituitary venous blood and resulted in an increase in the secretion of ACTH and alpha MSH in all horses. Furthermore, spontaneous AVP peaks occurred in pituitary venous blood between 90 and 180 min after AVP injection, indicating that the exogenous hormone did not suppress AVP secretion. oCRF administration led to a prolonged elevation in plasma CRF and an increase in secretion of ACTH and alpha MSH, but not AVP, in all horses. The pulsatile secretion of ACTH and alpha MSH was maintained despite plasma CRF levels in excess of 400 pmol/liter, and the timing of concentration changes in AVP and ACTH continued to be highly correlated. It is concluded that pulsatile ACTH secretion continues during cortisol, oCRF, or AVP administration. Like that of ACTH, alpha MSH secretion is stimulated by oCRF and AVP administration and suppressed by cortisol. Although the timing of concentration changes in ACTH and alpha MSH is highly correlated, the correlation of the actual concentrations of these two hormones varies considerably in different animals.

Effect of isolation stress on concentrations of arginine vasopressin, alpha-melanocyte-stimulating hormone and ACTH in the pituitary venous effluent of the normal horse.

A non-surgical, non-stressful technique was used for collection of pituitary venous blood from five conscious horses every minute for two 10-min periods before and during isolation from the herd, which caused a predictable, yet humane and physiological, emotional stress. Pituitary blood was also sampled every 5 min for two approximately 90-min periods before and after isolation, while jugular blood was sampled every 15 min throughout the experiment. During isolation, all horses became agitated, hyperventilating and sweating. Packed red cell volume increased, as did pituitary venous concentrations of adrenaline (mean +/- S.E.M. concentration before isolation, 621.5 +/- 112.3 pmol/l; peak during isolation, 2665.4 +/- 869.8 pmol/l; P less than 0.05) and noradrenaline (before, 871.8 +/- 111.8 pmol/l; peak, 2726.1 +/- 547.4 pmol/l; P less than 0.02). Concentrations of arginine vasopressin (AVP) were higher in pituitary venous but not in jugular blood during isolation than during the preceding 10-min period (P less than 0.05). Although AVP secretion increased in all horses, in three of the five it rose dramatically in the first minute of isolation to 25.7 (horse 1), 13.6 (horse 4) and 145.1 (horse 5) times the level in the last sample collected before isolation. Mean pituitary venous concentrations of ACTH and alpha-MSH increased during isolation in the three horses which had large increases in AVP secretion, but, overall, stress did not significantly affect ACTH or alpha-MSH secretion. Similarly, mean jugular cortisol levels were not significantly altered by isolation. However, the magnitudes of ACTH, AVP and alpha-MSH responses to isolation were negatively correlated with the jugular cortisol level before isolation. The changes in pituitary venous concentrations of ACTH and AVP were synchronous under resting conditions, whether samples were collected at intervals of 1 (P less than 0.01) or 5 (P less than 0.005) min; however, this synchrony was lost during isolation. The changes in pituitary venous concentrations of ACTH and alpha-MSH were synchronous both at rest (P less than 0.025 for 1-min sampling, P less than 0.01 for 5-min sampling) and during isolation (P less than 0.01). We conclude that isolation stress increases AVP secretion and may alter the temporal relationship between pituitary venous concentrations of AVP and ACTH. Furthermore, the magnitude of the responses of AVP, ACTH and alpha-MSH to isolation is significantly affected by the prevailing cortisol level.

A novel technique for measuring hypothalamic and pituitary hormone secretion rates from collection of pituitary venous effluent in the normal horse.

We have described a novel technique for collecting pituitary venous effluent in the horse by placing a cannula in the intercavernous sinus close to the outlet of the pituitary veins using a venous pathway unique to equids. Cannula placement and blood collection are carried out painlessly in fully conscious, ambulatory, unstressed animals. There is no interference to hypothalamic, pituitary or target organ function. The blood collected contains readily measurable concentrations of gonadotrophin-releasing hormone, and LH concentrations which can be up to 40 times those in concurrent peripheral blood samples. Four millilitre blood samples, a quantity which permits simultaneous measurement of many hypothalamic and pituitary hormones, can be collected at 2-min intervals for several days. Intercavernous sinus blood flow can be calculated allowing secretion rates of hypothalamic and pituitary hormones to be determined for any time-period. This model is uniquely useful for investigating the normal functional characteristics of several neuroendocrine and endocrine systems.

Spontaneous and stimulated adrenocorticotropin and vasopressin pulsatile secretion in the pituitary venous effluent of the horse.

Plasma ACTH, arginine vasopressin (AVP), and catecholamines were measured at 5-min intervals in the pituitary venous effluent of the unanesthetized horse. Pulses of ACTH and AVP were found to be surprisingly brief (usually of less than 10-min duration) and frequent (averaging between 15-25 min). A highly significant relationship in the changes in concentration of these two hormones was demonstrated (P less than 0.0002) both at rest and after a mild hypoglycemic stimulus. Although there was also a significant correlation (P less than 0.005) between simultaneous plasma ACTH and AVP values the pulse amplitude ratio of AVP to ACTH showed a considerable variation. A rise in cortisol appeared to have a greater suppressive effect on the amplitude of ACTH than AVP pulses. The gradient in hormonal concentration between pituitary effluent and jugular plasma was at times over 50-fold for ACTH, and 500-fold for AVP. A gradient was also found for epinephrine, norepinephrine, and dopamine. A highly significant correlation (P less than 0.005) was demonstrated between changes in norepinephrine, ACTH, and AVP concentrations, but no such relationship could be shown for epinephrine and dopamine. It is concluded that there is a close temporal relationship between changes in ACTH, AVP, and norepinephrine concentrations. Pulses of these hormones are greater in amplitude and more frequent than would have been suspected from sampling peripheral plasma. The variability in the pulse amplitude ratio of ACTH and AVP may suggest that other factors are affecting ACTH secretion. The ability to sample frequently for several hormones and to obtain a marked gradient in hormonal secretion between the pituitary venous effluent and jugular plasma suggest that the horse should provide an excellent animal model in which to study the regulation of hypothalamic and pituitary hormone secretion.