Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry.

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.

On-probe immunoaffinity extraction by matrix-assisted laser desorption/ionization mass spectrometry.

A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses.

Development and validation of two solid-phase enzyme immunoassays (ELISA) for quantitation of human epidermal growth factors (hEGFs).

PURPOSE: The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rat plasma. METHODS: The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. RESULTS: The calibration curves for hEGF1-48 and hEGF1-53 in plasma were validated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, respectively. Determined from replicate assays of hEGF1-48 quality control samples, the intra-assay precision and accuracy were < or = 8.8% RSD and within +/- 9.8%; and the inter-assay precision and accuracy were < or = 14.8% RSD and within +/- 9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality control samples, the intra-assay precision and accuracy were < or = 10.0% RSD and within +/- 8.5%; and the inter-assay precision and accuracy were < or = 10.0% RSD and within +/- 5.7% RE, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-53 assay using 200 microliters plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF alpha, TGF beta, PDGF, and FGF) or lymphokines (IL1 beta and TNF alpha). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. CONCLUSIONS: Two sensitive ELISA methods have been validated for quantitation of hEGF1-53 and hEGF1-48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentration of exogenous and endogenous epidermal growth factors.

Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

Behavioral and physiologic effects of CRH administered to infant primates undergoing maternal separation.

Infant rhesus monkeys briefly separated from their mothers emit frequent distress vocalizations and alter their activity levels. These behavioral alterations are accompanied by physiologic changes and activation of the hypothalamic-pituitary-adrenal axis. Because corticotropin-releasing hormone (CRH) is a major regulator of the pituitary-adrenal system and has been implicated as a mediator of other aspects of the stress response, we examined the effects of centrally and peripherally administered CRH on the infant monkey's separation response. Intracerebroventricular (ICV) doses less than 10 micrograms did not alter behavior; 10-micrograms doses inhibited behavior without affecting distress vocalizations. The behavioral inhibition did not appear to be due to nonspecific sedation and may be related to increased fearfulness. It was accompanied by increases in adrenocorticotropic hormone (ACTH) and cortisol and small but significant decreases in body temperature and mean arterial pressure. Within an individual animal, no relationship was seen between ICV CRH's effects on physiologic parameters and its effects on behavior. ICV CRH produced significant increases in plasma concentrations of CRH, suggesting the possibility that the effects of CRH were mediated through peripheral mechanisms. However, 10 micrograms of CRH administered intravenously (IV) had no effect on behavior, blood pressure, or body temperature, nor did it increase plasma concentrations of ACTH or cortisol beyond the expected separation-induced elevation. This is interesting because plasma levels of CRH after IV CRH were much greater than after ICV CRH. Our findings suggest that in infant rhesus monkeys undergoing maternal separation, brain CRH systems mediate behavioral inhibition and pituitary-adrenal activation.

Mood state and salivary cortisol levels following overtraining in female swimmers.

Mood, as measured by the Profile of Mood States questionnaire, and resting salivary cortisol levels were examined in 14 female college swimmers during progressive increases and decreases in training volume, and were compared to the same measures in eight active college women who served as controls. Training volume increased from 2,000 yards/day in September (baseline) to a peak of 12,000 yards/day in January (overtraining), followed by a reduction in training (taper) to 4,500 yards/day by February. The swimmers experienced significant (p less than 0.01) alterations in tension, depression, anger, vigor, fatigue and global mood across the training season compared to the controls. Salivary cortisol was significantly (p less than 0.01) greater in the swimmers compared to the controls during baseline and overtraining, but was not different between the groups following the taper. Salivary cortisol was significantly correlated with depressed mood during overtraining (r = .50; p less than 0.05) but not at baseline or taper. Global mood, depression, and salivary cortisol were significantly (p less than 0.05) higher during the overtraining phase in those swimmers classified as stale, compared to those swimmers who did not exhibit large performance decrements.

Pulsatile ACTH secretion: variation with time of day and relationship to cortisol.

It has long been known that ACTH is secreted in an episodic fashion demonstrating circadian and ultradian rhythms. High intensity venous sampling has recently revealed that in addition to these larger ultradian fluctuations in hormone levels, plasma ACTH in rats demonstrates high frequency, low amplitude oscillations which have been called "micropulses." These micropulses were not detected in previous studies due to sampling intervals of greater than 5 minutes. To investigate the presence of these ACTH micropulses in a primate species, blood samples were drawn from six chair-restrained rhesus monkeys at one-minute intervals for up to 70 minutes and plasma was assayed for immunoreactive ACTH. To assess the variation in ACTH micropulse parameters with time of day and the relationship to cortisol secretion, four of the monkeys were sampled for three 70-minute periods beginning at 0530, 1100, and 1730 hours, and plasma was assayed for immunoreactive ACTH and cortisol. Analysis of the data revealed that ACTH and cortisol are secreted in micropulses in rhesus monkeys with marked individual variation in the pattern of secretion and a concurrence of approximately 75% of ACTH and cortisol micropulses. Difference in pulse amplitude but not frequency appeared to contribute to the circadian variation in mean ACTH levels and a sampling interval of two minutes appeared to be adequate for accurately identifying micropulses of ACTH.

Opiate modulation of separation-induced distress in non-human primates.

Infant rhesus monkeys respond to separation from their mothers with a dramatic increase in vocalizations and activation of autonomic and pituitary-adrenal systems. Using the mother-infant separation paradigm in rhesus monkeys, we focused on the role of opiate systems in modulating the behavioral and neuroendocrine consequences of a brief, naturally occurring stressor. In the first experiment, morphine 0.1 mg/kg significantly decreased separation-induced vocalizations without affecting activity levels. In the second experiment, naloxone 1.0 mg/kg increased distress vocalizations but lower doses had no effect. In the third experiment we blocked the effect of morphine 0.1 mg/kg with naloxone 0.1 mg/kg, a dose of naloxone that had no intrinsic effects of its own. This suggests that the reduction of separation-induced vocalizations by morphine is mediated by opiate receptors. The last experiment demonstrated that separation-induced increases in pituitary-adrenal hormones can also be modulated by opiate agonists and antagonists. These findings are consistent with work in non-primate species and support the hypothesis that opiate receptors are specifically involved in mediating separation-induced vocalizations and pituitary-adrenal activation in primates.

Stressor controllability during pregnancy influences pituitary-adrenal hormone concentrations and analgesic responsiveness in offspring.

Repeated escapable shock, yoked-inescapable shock, or no-shock treatments were administered to female rats before parturition to investigate the effects of stressor controllability on offspring pituitary-adrenal hormone concentrations and stress-induced analgesic reactions. Female rats exposed to escapable shock treatments received tail-shock in boxes containing a wheel that allowed shocks to be terminated after rotation. Rats in the yoked-inescapable shock group received an identical amount and pattern of tail-shock. However, shock was terminated only after wheel rotation by the rat undergoing escapable shock treatments. Female rats in the no-shock group were simply placed in wheel-turn boxes. Fourteen-day-old offspring were exposed for 10-min to either a separation-stress or shock-induced stress test. The former test consisted of separating and isolating the pup from the mother and siblings, whereas the latter involved the administration of five brief, 1.0 sec, low intensity, 0.5 mA, foot-shocks. Immediately after exposure to foot-shocks, pups were given a tail-flick test to assess their analgesic response. Plasma was obtained from pups immediately after separation and tail-flick tests and ACTH and corticosterone concentrations were assayed by radioimmunoassay. Results indicated that prenatal inescapable shock treatments resulted in offspring with significantly higher plasma ACTH and corticosterone concentrations than offspring exposed to prenatal escapable shock or no-shock treatments. Offspring of females exposed to inescapable shock also exhibited greater increases from basal concentrations in ACTH and corticosterone after stress. Furthermore, prenatal escapable and inescapable shock treatments significantly altered shock-induced analgesic thresholds.(ABSTRACT TRUNCATED AT 250 WORDS)

A diurnal rhythm in cerebrospinal fluid corticotrophin-releasing hormone different from the rhythm of pituitary-adrenal activity.

Corticotrophin-releasing hormone (CRH) appears to be involved in the pathophysiology of various neuropsychiatric illnesses. Because of the potential importance of determining CRH concentrations in cerebrospinal fluid (CSF) in humans and the constraints on human experimentation, we used a rhesus monkey model to study factors affecting CSF-CRH concentrations and the association between CRH concentrations and changes in plasma ACTH and cortisol levels. CSF-CRH concentrations followed a diurnal rhythm not closely linked to that of the anterior pituitary-adrenal system. Manipulations that increased release of pituitary ACTH did not affect CSF-CRH concentrations. Our data show that sampling time should be controlled in human CSF-CRH studies and suggest that altered CSF-CRH levels reflect dysregulation of extrahypothalamic CRH neurons.

Rise in plasma beta-endorphin, ACTH, and cortisol in cancer patients undergoing whole body hyperthermia.

It has been previously reported that sauna-induced fevers (approximately 39 degrees C) result in rises of beta-endorphins in normal volunteers. This report describes changes in plasma beta-endorphins in cancer patients undergoing whole body hyperthermia (40.5 degrees C to 41.8 degrees C). Results presented show that there is a linear relationship between thermal stress, defined in terms of core temperature and/or duration of hyperthermia, and the quantitative rise in plasma beta-endorphin levels. Data relating to changes in ACTH and cortisol levels are in a single temperature range (41.5 degrees C--41.8 degrees C) are also reported.

Separation distress in infant rhesus monkeys: effects of diazepam and Ro 15-1788.

Disruption of the primate mother-infant attachment bond is a naturally occurring stressor that results in marked behavioral, physiological, and endocrine activation. We studied the effect that altering benzodiazepine systems has on the behavioral and endocrine response of infant rhesus monkeys (1-27 weeks of age) to brief separation from their mothers. In the first experiment, the benzodiazepine agonist diazepam (0.1 and 1.0 mg/kg) significantly increased locomotion and social behavior and decreased inactivity and distress vocalizations in infant monkeys undergoing separation. In the second experiment, the benzodiazepine antagonist Ro 15-1788 (5 and 10 mg/kg) had no significant effects on the infants' separation response. In the third experiment, administration of diazepam 1.0 mg/kg was followed by administration of Ro 15-1788 10 mg/kg in infants undergoing separation. Ro 15-1788 blocked the decreases both in inactivity and in plasma ACTH and cortisol concentrations caused by diazepam, suggesting that these effects are mediated through benzodiazepine receptors. These data support the hypothesis that in primates, endogenous benzodiazepine systems modulate the behavioral and endocrine response to the naturally occurring stress of separation.

Neuroendocrine changes in patients undergoing whole body hyperthermia.

A phase I study of whole body hyperthermia (WBH) (52 treatments/12 patients) utilizing a radiant heat device has been completed. This study incorporated a temperature escalation scheme from 39.5 to 41.8 degrees C for up to 150 min. Pain relief or a sense of well being was observed post-WBH in the first three patients entered in this study. We postulated that WBH might result in increases in the opiate peptide beta-endorphin. Therefore we elected to study prospectively the next six patients entered in this study to test the hypothesis that WBH stimulates the neuroendocrine axis. Results are reported which show thermal-induced increases in plasma levels of beta-endorphin as well as prolactin, ACTH and cortisol.

Effects of dexamethasone on central and peripheral ACTH systems in the rat.

To investigate the simultaneous effects of dexamethasone on peripheral and central adrenocorticotropic hormone (ACTH) systems, rats were treated with dexamethasone or saline for 4 days. Pituitary, plasma, hypothalamus and cerebrospinal fluid (CSF) were then collected and analyzed for ACTH immunoreactivity. Additionally, hypothalamic tissue extracts were analyzed for corticotropin-releasing hormone (CRH) immunoreactivity. Dexamethasone significantly lowered peripheral levels of ACTH as measured in pituitary and plasma. Hypothalamic ACTH content significantly increased while CSF ACTH significantly decreased with dexamethasone treatment. Hypothalamic CRH concentrations showed a small but statistically insignificant decrease. These results suggest that prolonged exposure to dexamethasone affects central as well as peripheral ACTH activity, corroborate our previous findings in rhesus monkeys of decreased CSF ACTH in response to prolonged dexamethasone treatment, suggest that dexamethasone may inhibit the release of ACTH from hypothalamic neurons into the CSF, and provide evidence that the effect of dexamethasone on pituitary ACTH content is of greater magnitude than its effect on hypothalamic CRH.

Stressor controllability and the pituitary-adrenal system.

Stressor controllability can alter both behavior and pituitary-adrenal activity. Potential mediation of these behavioral effects by differential pituitary-adrenal output requires that the precise conditions that lead to differential behavioral consequences also produce differential pituitary-adrenal activity. Both plasma ACTH and corticosterone levels were measured at various times following escapable and yoked inescapable electric shock conditions known to produce differential behavioral outcomes. The escapable and inescapable shock procedures did not produce a detectable differential effect. Both shock conditions produced equivalent elevation of ACTH and corticosterone. Neither decay rates nor the ACTH and corticosterone response to shock reexposure differed among shocked groups.

Episodic secretion of ACTH in rats.

While the circadian rhythm of pituitary adrenocorticotropin (ACTH) secretion has been well characterized, the ultradian rhythm has been less thoroughly investigated. To study the episodic nature of ACTH secretion, unrestrained, unanesthetized rats were bled continuously through indwelling jugular venous cannulae and blood sampled for up to 75 minutes at one-minute intervals beginning at 1100 hr (n=6) or 1730 hr (n=4). Sporadic low-amplitude micropulses were observed at both times of day. In addition, infrequent "superpulses" were observed in the evening. Analysis of pulse parameters revealed a significant (p less than 0.001) difference in pulse amplitude but no difference in pulse frequency of interpeak interval between morning and evening. As with other episodically secreted hormones, the threshold for pulse identification and the sampling interval were found to influence the observed pulse parameters.

A rostral-caudal concentration gradient in cerebrospinal fluid adrenocorticotropin.

In two separate experiments, cerebrospinal fluid (CSF) adrenocorticotropin immunoreactive (ACTH-IR) concentrations in the rhesus monkey followed a significant rostral-caudal gradient. In the first study, CSF was sampled from an indwelling catheter in awake animals. The mean ACTH-IR concentration in the cisternal region was 12.3 pg/ml, as compared to 8.56 pg/ml in the lumbar region. In the second study, CSF was sampled in a different group of monkeys by percutaneous puncture at the cisterna magna and at L5-L6. In this study, the mean ACTH-IR concentration in samples collected from the cistern was also greater than the concentration from L5-L6. In addition, a significant correlation within subjects was found between samples collected from the two sites (r = 0.86). These results demonstrate that the site of CSF sampling is a variable in determining CSF ACTH-IR concentrations and suggest that lumbar CSF ACTH-IR concentrations in humans may be interpreted as indexes of ACTH changes at higher levels in the central nervous system.

Behavioral stress decreases plasma oxytocin concentrations in primates.

Using rhesus monkeys, we studied the effects of a behavioral stress on plasma concentrations of adrenocorticotropin, oxytocin, and vasopressin. The stress resulted in significant increases in adrenocorticotropin and significant decreases in oxytocin concentrations. No significant changes were seen in vasopressin concentrations. To further explore the relationship between plasma oxytocin and pituitary-adrenal function, dexamethasone was administered to rhesus monkeys. This resulted in significant increases in plasma oxytocin concentrations, while adrenocorticotropin decreased.

The diurnal variation of immunoreactive adrenocorticotropin in rhesus monkey plasma and cerebrospinal fluid.

ACTH immunoreactivity (ACTH-IR) in the plasma and cerebrospinal fluid (CSF) collected simultaneously from rhesus monkeys was found to undergo significant diurnal variations. In plasma, the mean peak ACTH-IR was 15.4 +/- 1.95 pg/ml at 0500 h, and the mean minimum concentration was 9.05 +/- 1.80 pg/ml at 1800 h. In CSF, the mean peak ACTH-IR concentration occurred at 1900 h and was 4.64 +/- 0.41 pg/ml. The mean minimum CSF ACTH-IR concentration was 2.93 +/- 0.26 pg/ml, occurring at 0500 h. This is the first report of a diurnal variation in CSF ACTH-IR concentration and is consistent with other work suggesting that plasma ACTH and CSF ACTH originate from different sources.

Effects of acute behavioral stress on plasma and cerebrospinal fluid ACTH and beta-endorphin in rhesus monkeys.

To elucidate the effect of acute behavioral stress on plasma and cerebrospinal fluid (CSF) concentrations of adrenocorticotropic hormone (ACTH) and beta-endorphin, rhesus monkeys were subjected to 30 min of confinement stress. Simultaneous plasma and CSF samples revealed no significant change in CSF ACTH or beta-endorphin up to 120 min after the onset of the stress despite significant elevations in plasma cortisol, ACTH, and beta-endorphin. It is suggested that acute behavioral stress does not alter CSF ACTH or beta-endorphin, and that this information may be clinically useful for future human studies of CSF ACTH and beta-endorphin in neuropsychiatric illnesses.