Determination Of Cortisol In Plasma Research Articles

Effects of Autogenic Training on Nitroglycerin‐Induced Headaches

Objectives.—To investigate the prophylactic and acute effects of autogenic training (AT) during a nitroglycerin‐induced migraine attack.Methods.—Thirty female migraineurs (without aura) and 11 controls participated in the study. Of these, 11 migraineurs and 5 controls practiced AT regularly for at least 6 months prior to and during the sublingual nitroglycerin test. Headache intensity and characteristics were recorded with a standardized method. During the nitroglycerin challenge, blood was collected for plasma cortisol determination and blood pressure and pulse rate were recorded.Results.—As a long‐term preventive treatment, AT significantly decreased the mean headache frequency and intensity (P= .001) compared to the pretreatment period in the migraineurs who regularly practiced AT (n = 11). During the nitroglycerin challenge, AT successfully attenuated the nitroglycerin‐induced acute decrease in blood pressure and pulse rate (P= .013; n = 16 AT subjects vs n = 25 non‐AT subjects). However, it was not effective in preventing immediate headache (P= .71), did not decrease the frequency of acute migraine attacks (P= .79), and could not alleviate acute migraine pain (P= .78; n = 16 AT subjects vs n = 25 non‐AT subjects). Plasma cortisol concentration significantly increased (P= .003) during the acute migraine attack (n = 22), and migraine intensity correlated with plasma cortisol elevations (P < .001; n = 41) and showed a tendency of negative correlation with morning plasma cortisol concentration (P= .08; n = 41). However, AT did not alter plasma cortisol responses (P= .99; n = 16 AT subjects vs n = 25 non‐AT subjects).Conclusion.—(1) The long‐term AT therapy proved to be a significantly effective preventive intervention in migraine sufferers. We hypothesized that this long‐term effect of AT is based on modulation of the pain anticipation system, which is strongly correlated with function of the anterior cingulate cortex. (2) We demonstrated that AT could not alter the nitroglycerin‐induced acute migraine attacks, which are directly related to the dysfunctional brainstem activation according to previous studies. (3) Our results suggested that there are multiple, complex relationships between cortisol responses and migraine pain, which are possibly mediated by the brain serotonergic system. (4) In addition, our results provide further evidence that nitroglycerin‐induced vasodilatation is not directly connected to either immediate headache or delayed migraine attack.

The use of intravenous catheterisation with a rest period is useful for determination of plasma cortisol levels but not plasma prolactin levels

Objective The use of an intravenous catheter with a rest period has been recommended to avoid false-positive results for hyperprolactinaemia and false-negative results for hypocortisolaemia. We tested the relevance of this recommendation. Design Plasma cortisol and prolactin levels were determined before (T-15) and after a 15-min rest period (T0) in 119 patients, 38 males (M) and 81 females (F). 52 of the 119 patients were known (K; 30 females and 22 males) and 67 unknown (UK; 49 females and 18 males) to the unit. Results Prolactin was lower after rest in women (12.3 ± 22.7 ng/l vs 11.7 ± 22.5 ng/ml, P = 0.03), but not in men (6.2 ± 4.5 ng/ml at T-15 vs 5.8 ± 3.2 ng/ml at T0, P = 0.09), in the UK subgroup (10.6 ± 20.7 ng/ml at T-15 vs 10.1 ± 20.9 ng/ml at T0, P = 0.06) and in the K subgroup (10.1 ± 16.7 ng/ml at T-15 vs 9.7 ± 15.8 ng/ml at T0, P = 0.08). None of the patients with prolactin levels higher than 20 ng/ml at T-15 diminished its prolactin value below this cut-off value. Plasma cortisol levels were lower after rest in women (17.9 ± 5.9 μg/dl at T-15 vs 16.5 ± 6.1 μg/dl at T0, P < 0.0001), in the UK subgroup (18 ± 6.1 μg/dl at T-15 vs 16.6 ± 6.4 μg/dl at T0, P = 0.0003) but not in men (18 ± 4.4 μg/dl at T-15 vs 17.5 ± 5.8 μg/dl at T0, P = 0.47) and in the K subgroup (17.8 ± 4.6 μg/dl at T-15 vs 17 ± 5.4 μg/dl at T0, P = 0.13). At T0, 3.3% and 15% of patients presented values below the cut-off value of 10 μg/dl (276 nmol/l) and 17 μg/dl (470 nmol/l), respectively. Conclusion These results don't justify intravenous catheterisation with a rest period for plasma prolactin determination in contrast with plasma cortisol determination.

Simultaneous Determination of Plasma Cortisol by High Pressure Liquid Chromatography and Radioimmunoassay Methods in Fish

The aim of the study was to compare the sensitivity of High Performance Liquid Chromatography (HPLC) and Radioimmunoassay (RIA) analytical methods in the determination of blood plasma cortisol level of common carp (Cyprinus carpio L.). Cortisol, the most potent glucocorticoid, is widely used as an indicator of stress. The monitoring of plasma cortisol concentration was performed during the test focused on stress response caused by handling and long-distance transport of common carp. The samples were collected before, during and after transport. The correlation between blood cortisol level in fish measured by HPLC and RIA was determined using 66 samples. The correlation was HPLC = 0.9454 RIA + 0.40676 and correlation coefficient was 0.815. From the presented results it can be concluded that both methods (RIA and HPLC) can be used for the determination of plasma (serum) cortisol level.

Reducing mother-young separation distress by inducing ewes into oestrus at the day of weaning

AbstractThe aim of this study was to determine whether or not oestrus induction on the day of weaning would reduce the distress experienced by ewes upon separation from lambs. For this, 43 ewes, their eight week-old lambs and six mature rams were used. Prior to weaning, 21 of these ewes were induced to display oestrus on the day of mother-young separation (treated group [T]) while the remaining 22 untreated ewes served as controls (C). T and C ewes were housed together. Blood samples were collected on the day of weaning (prior to mother-young separation and 24 hours later) and then nine days later, for plasma progesterone (P4) and cortisol determination. Lambs were separated from their dams by a wire fence at weaning in full view of each other. Three rams were tethered to posts with 3 m plastic chains in the ewes' pen. T and C groups were simultaneously tested in identical test pens. C ewes showed a greater increase in serum cortisol concentration after separation from lambs (47.64 ± 4.26 n mol l–1) than T ewes (28.79 ± 6.29 n mol l–1). T ewes exhibited fewer vocalisations ewe–1h–1 and fewer vocalisations at 6, 12 and 18 h post separation than C ewes. On the day of weaning, more T ewes were seen to be situated away from lambs and in close proximity to the males, compared to the C group. Thus, more T than C ewes were mounted (14 as opposed to 3) and these received a greater number of mounts (1.02 ± 0.23 per hour, as opposed to 0.11 ± 0.06). It was concluded that by inducing ewes into oestrus at weaning it is possible to reduce the signs of separation distress.

Simultaneous determination of cortisol and prednisolone in body fluids by using HPLC–DAD coupled with second-order calibration based on alternating trilinear decomposition

A novel method for simultaneous determination of cortisol and prednisolone in body fluids has been developed in this paper. Three-way data recorded by high-performance liquid chromatography with a diode array detector (HPLC-DAD) have been analyzed by second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm. The chemometric methodology selected exploits the second-order advantage of the three-way data arrays, which allows one to obtain concentrations of individual calibrated analytes even in the presence of interferences not present in the calibration samples (e.g. background in urine or plasma). It was applied to simultaneous determination of cortisol and prednisolone in both plasma and urine samples. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of the analytes could be obtained with HPLC-DAD coupled with second-order calibration based on ATLD. Sample preparation was based on solvent extraction (SE), and quantification can be carried out with simple mobile phase. The time required for the quantification process is shorter than other methods.

Simultaneous determination of 6β-hydroxycortisol and cortisol in human urine and plasma by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity

This study describes an high-performance liquid chromatographic (HPLC)-UV method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol (F) in human urine and plasma. The within-day relative standard deviation of the three concentrations for both analytes was less than 6.2%. Accuracy determined at three concentrations ranged between 95 and 107%. The extraction recoveries were 64.1+/-4.3 and 88.1+/-2.4% at three concentrations for 6beta-OHF and F in urine, respectively. The extraction recoveries were 88.7+/-1.4% at three concentrations for F in plasma. This is the first HPLC method that can simultaneously determine 6beta-OHF and F in human urine and plasma and is suitable for routine assessment of the CYP3A activity expressed as 6beta-hydroxylation clearance.

Lymphocyte functions in dairy cows in hot environment

This study was carried out to ascertain the effects of intense high environmental temperatures (HET) on lymphocyte functions in periparturient dairy cows. The study was undertaken from the beginning of March through the end of July 2003 in a commercial dairy unit located approximately 40 km north of Rome. Thirty-four Holstein cows were utilised in the study. Twenty-two of these cows gave birth in spring (SP cows), from 28 March to 30 April. The remaining 12 cows gave birth in summer (SU cows), between 15 June and 2 July. The two groups of cows were balanced for parity and were fed the same rations. Blood samples were taken 4, 3, 2 and 1 week before calving, and 1, 2 and 4 weeks after calving, in order to evaluate peripheral blood mononuclear cell (PBMC) function in vitro, and to determine plasma cortisol concentrations. After isolation, the PBMC were stimulated with mitogens and their response in terms of DNA synthesis and IgM secretion was measured. During spring, either the day (9-20 h) or the night (21-8 h) temperature humidity index (THI) was below the upper critical THI (72) established for dairy cows. During summer, the mean daily THI values were 79.5+/-2.9 during the day and 70.1+/-4.7 during the night. Furthermore, during summer, three heat waves (a period of at least 3 consecutive days during which there were less than 10 recovery hours) occurred. Recovery hours were intended hours with a THI below 72. The first heat wave lasted 5 days, the second 6 days, and the third 15 days. Compared to the SP cows, over the entire periparturient period the extent of DNA synthesis and IgM secretion levels were lower (P ranging from <0.01 to 0.0001) and higher (P<0.01) respectively, in the SU cows. Before calving, the SU cows also presented higher (P<0.01) concentrations of plasma cortisol compared to the SP cows. This study indicates that the effects of HET on the immune response depend on the specific immune function under consideration, and that neuroendocrinal changes due to HET may play a role in the perturbation of immune functions.

Rearing environment and hypothalamic-pituitary-adrenal regulation in young rhesus monkeys (Macaca mulatta)

A mammal's early social environment has important regulatory effects on its behavior and physiology, and this is especially true for regulation of the hypothalamic-pituitary-adrenal (HPA) system. The present study was designed to test hypotheses that various aspects of the social environment are important influences on HPA regulation. Seven hundred seventy eight, 3- to 4-month-old rhesus monkeys were studied as part of a standardized, 24-hr biobehavioral assessment program, which included blood sampling to determine plasma cortisol concentrations. Results indicate that nursery-rearing results in a reduced cortisol set-point for the HPA system, and, for nursery-reared (NR) animals, more peer exposure during infancy is associated with a higher set-point. Age and sex differences during this period were evident but small in magnitude. These data demonstrate the important regulatory role of the social environment on nonhuman primate physiology and suggest caution in assuming that differences between individuals' cortisol levels reflect only differences in perceptions of the "stressfulness" of events.

A dose-response study of salivary cortisol after dexamethasone suppression test in Cushing's disease and its potential use in the differential diagnosis of Cushing's syndrome.

A dose-response study with different doses of dexamethasone (dex) to assess the corticotrophic resistance in Cushing's disease (CD) using salivary cortisol as an end point has not yet been evaluated. We also reported our experience with salivary cortisol compared to plasma cortisol determination during dex suppression test (DST) and after ovine corticotrophin release hormone (oCRH) test in the differential diagnosis of Cushing's syndrome (CS). We studied 46 patients with CS, including 28 patients with CD, 16 with adrenal disease and two with occult ectopic adrenocorticotropic hormone (ACTH) tumours. Salivary cortisol was compared to plasma cortisol and ACTH during a DST 2 mg for 2 days, 8 mg for 2 days and 24 mg for 1 day, and after oCRH test. We observed a dose-dependent suppression of salivary cortisol, plasma cortisol and ACTH in CD patients. Salivary cortisol presented a higher percentage of suppression than plasma cortisol: 42% vs. 15% (P < 0.002), 82% vs. 67% (P < 0.002) and 90% vs. 83% (P < 0.03) after 2, 8 and 24 mg/day dex, respectively. The lowest percentage of suppression was observed for plasma ACTH. The parallelism of these lines identified that the criterion of 65% suppression of salivary cortisol corresponding to 50% suppression of plasma cortisol after 8 mg/day for 2 days is consistent with CD. The sensitivity and specificity using 50% suppression for plasma cortisol were 81% and 83%, respectively, for 8 mg DST. Using the criterion of 65% suppression of salivary cortisol, the sensitivity and specificity were 86% and 100%, respectively, for 8 mg DST. After oCRH test the sensitivity and specificity were 86% and 91%, respectively, for ACTH, 100% and 64%, respectively, for plasma cortisol and 93% and 91%, respectively, (20% of increment) or 86% and 100%, respectively, (35% increment) for salivary cortisol. In conclusion, salivary cortisol presents more profound suppression than plasma cortisol or ACTH in a dose-response pattern after different doses of dex in patients with CD. In addition, our data suggest that measurement of salivary cortisol might improve the DST as compared to plasma cortisol in the differential diagnosis of CS.

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis ® hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 μm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.

Effects of intracerebroventricular infusions of corticotropin‐releasing hormone in sheep

ABSTRACTTo provide new insights into the neural mechanisms of physiological and behavioral responses to stressors in sheep, acute changes in endocrine, autonomic and behavioral functions following 30 min infusions of ovine‐corticotropin‐releasing hormone (oCRH; 0, 0.5, 5 or 50 µg/0.5 mL of artificial cerebrospinal fluid/30 min) into the third ventricle of sheep (n = 7–8) were examined. Serial blood samples were collected through indwelling jugular catheters to determine plasma cortisol concentrations (CORT). Heart rate (HR) and rectal temperature (RT) were obtained via telemetry systems. The behaviors of the animal were monitored simultaneously. Intracerebroventricular infusions of CRH dose‐dependently induced an increase in CORT; there was a time–treatment interaction in CORT (P &lt; 0.001). There was not a time–treatment interaction either in HR (P = 0.29) or in RT (P = 0.28). That RT showed a tendency to decrease with higher doses of CRH in sheep was in contradiction to previous reports in rats and pigs. As to changes in behavioral function, only the induction of bleating was marked. These results suggest that in physiological and behavioral responses of sheep to stressors, CRH regulates the increase in CORT and the induction of bleating. However, CRH might have little function in sympathetic nervous activation during physiological responses to stressors in sheep.

Cortisol and dehydroepiandosterone sulfate plasma levels and their relationship to aging, cognitive function, and dementia

We have studied the relationship between dehydroepiandrosterone sulfate (DHEAS), cortisol, and cognitive function in a population of demented patients ( n=29), age-matched controls ( n=46), and younger subjects ( n=11). All were submitted to morning collection of blood for determination of plasma cortisol and DHEAS measured by 125I radioimmunoassay. DHEAS levels and cortisol/DHEAS ratios were significantly different among groups with higher DHEAS levels and lower cortisol/DHEAS ratios in younger people (Bonferroni p<.05). Cortisol levels were associated to the presence of dementia (Odds ratio=.93; 95% CI, .86–1.01). There was no difference between DHEAS levels of demented and age-matched controls; however, demented patients showed a trend for higher cortisol/DHEAS ratios than age-matched controls and the latter showed higher ratio values than younger subjects. DHEAS and cortisol plasma values were significantly correlated in all individuals ( p<.01). In this study cortisol was independently associated to the presence of dementia.

The cortisol and glucocorticoid receptor response to low dose dexamethasone administration in aging combat veterans and holocaust survivors with and without posttraumatic stress disorder

Background: Because alterations in cortisol negative feedback inhibition associated with aging are generally opposite of those observed in posttraumatic stress disorder (PTSD), we examined the cortisol and glucocorticoid receptor (GR) response to dexamethasone (DEX) in older trauma survivors. Methods: Twenty-three Holocaust survivors (9 men, 14 women), 27 combat veterans (all male), and 10 comparison subjects (7 men, 3 women) provided samples for plasma or salivary cortisol and glucocorticoid receptor determination in mononuclear leukocytes at 8:00 am on the day of, and following, 0.5 mg of DEX at 11:00 pm. Results: Greater percent suppression of cortisol and lymphocyte GR was observed in older trauma survivors with PTSD compared to survivors without PTSD and comparison subjects. There was a significant main effect of depression in the direction of reduced suppression following DEX, consistent with the effects of DEX in major depressive disorder patients. Responses to DEX were uncorrelated with PTSD symptom severity, but cortisol suppression was associated with years elapsed since the most recent, but not focal, traumatic event. Conclusions: The response to DEX is generally similar in older and younger trauma survivors, but the findings suggest that age, symptom severity, and lifetime trauma exposure characteristics may influence this response.

Timing of Initiation of the Preovulatory Luteinizing Hormone Surge and Its Relationship with the Circadian Cortisol Rhythm in the Human

The relationship between the hypothalamo-pituitary-gonadal (HPG) axis and the hypothalamo-pituitary-adrenal (HPA) axis has been well documented in the rat. In most cases, a negative coupling was observed and an inhibitory effect of the HPA axis upon the HPG was shown. In the female rat, a marked circadian rhythm of corticosterone plasma values is observed during each day of the estrous cycle, with maximal values around 08:00 p.m. The preovulatory luteinizing hormone (LH) surge also occurs at 08:00 p.m. on the day of proestrus. Here we measured circadian variations of plasma cortisol in humans in relation with the time of initiation of the preovulatory LH surge. Blood samples were taken at 08:00 a.m., 12:00 a.m., 04:00 p.m., 08:00 p.m., 12:00 p.m., and 04:00 a.m. from 19 subjects for 4 consecutive days, once 17β-estradiol (E₂) values reached 125 pg/ml (days 7–10 of the menstrual cycle). Serum E₂ and LH determinations were performed by microparticle enzyme immunoassays. Serum progesterone and plasma cortisol determinations were made using RIA methods. For plasma cortisol values, a marked circadian rhythm, with 2- to 3-fold higher values during the morning than during the afternoon, was almost identical before, during and after the LH surge. However, values were generally higher during the follicular phase than during the luteal phase. Maximum cortisol values occurred between 04:00 and 08:00 a.m. and minimal cortisol values between 04:00 and 08:00 p.m. Initiation of the LH surge (50% over the mean of previous values) occurred at 04:00 a.m. (20% of the cases) or at 08:00 a.m. (80% of the cases). There was a strong coupling between the onset of the surge and the acrophase of the cortisol circadian rhythm: maximal cortisol plasma values were seen at 04:00 a.m. when the LH preovulatory surge started at 04:00 a.m. and 08:00 a.m. when it started at 08:00 a.m. The present results show that the positive coupling documented in the female rat between the HPA and the HPG axis at the time of preovulatory LH surge is also present during the menstrual cycle in the human.

The effect of confinement on motivation to exercise in young dairy calves

The objective of this research was to determine if confinement of 8-day-old calves for varying lengths of time is associated with an increase in motivation to perform locomotor behaviors. Holstein heifer and bull calves ( N=48) were used in a factorial arrangement with two crossed factors. Factor A was housing with two levels (individual confinement versus group pens) and factor B was hours in confinement with four levels (6, 12, 24, and 48 h). Individual confinement was in 1.06 m×1.06 m pens, while group pens had a 3.68 m×6.09 m outside run and a 3.68 m×6.09 m covered area that also contained a 3.68 m×2.44 m area bedded with wood shavings. The calves were placed on treatment when they were 8±2 days of age. At the end of the treatments, a blood sample was taken for plasma cortisol determination and lymphocyte counts and the calves were open-field tested for 5 min. Walk, trot, distance traveled and behaviors performed while standing during the open-field test were higher in the calves kept in group pens ( P=0.0003, 0.01, 0.04 and 0.04), but were not influenced by hours in treatment. Calves confined for 48 h had greater incidences of kicking and falling ( P=0.014 and 0.025). Lymphocyte count ( P=0.029) was lower in the calves confined for 12 h, but there was not a trend across hours in confinement that indicated a consistent effect. Housing or hours in treatment did not affect canter, buck, buck–kick, rear, stumble, vocalization and cortisol concentrations. The interaction between hours in treatment and housing was not significant for any of the variables tested. This study suggests that 2 days may not have been enough time for the effects of close confinement to influence motivation in young calves, or that calves averaging 8 days of age may be too young to display increased motivation for locomotor activity. Confinement of such young calves actually inhibits locomotor activity in open-field tests.

Administration of ACTH to restrained, pregnant sows alters their pigs' hypothalamic-pituitary-adrenal (HPA) axis.

This study was designed to examine the physiology and behavior of pigs whose dams were snared and then injected with ACTH during gestation. Administration of ACTH to dams during pregnancy has been shown to replicate the effects of prenatal stress in other species. Control sows (n = 8) were given no treatment, whereas the treatment sows (ACTH, n = 8) were immobilized by snaring the snout and then administered an i.v. injection of ACTH (1 IU/kg BW) weekly from 6 to 12 wk of gestation. A pig was killed from each sow at 1, 30, and 60 d of age. The hypothalamus, pituitary gland, adrenal glands, and liver were immediately obtained to determine the amounts of corticotropin-releasing hormone (CRH), beta-endorphin, and mRNA for pro-opiomelanocorticotropin (POMC), the ACTH receptor (ACTH-R), and insulin-like growth factor I (IGF-I). Pituitary corticotrope and somatotrope cell numbers and adrenal cortex-to-medulla area ratios (CORT:MED) were also determined. Pigs' behaviors were recorded at 6 and 8 wk of age. At 75 d of age, a blood sample was taken and a biopsy puncture was created on one pig from each litter, then pigs were stressed by mixing. Blood was sampled every other day for 10 d to determine plasma cortisol concentrations and differential leukocyte counts. Biopsy damage was evaluated for healing. At 1 d of age, control pigs tended to weigh more (P = .09), have a lower expression of ACTH-R mRNA (P = .01) and IGF-I mRNA (P = .01), and a lower CORT:MED (P = .04) than ACTH pigs. At 30 d of age, control pigs had a greater concentration of beta-endorphin (P = .01) and tended to have a lower concentration of CRH (P = .09) and IGF-I mRNA (P = .10) than ACTH pigs. At 60 d of age, control pigs tended to have lighter pituitary glands (P = .08), a lower expression of POMC mRNA (P = .02), and a CORT:MED (P = .003) than ACTH pigs. At 8 wk of age, control pigs performed a higher frequency of belly nosing (P = .07) and oral vice behaviors (P = .01) than ACTH pigs. In response to mixing stress, control pigs had lesser concentrations of plasma cortisol (P = .03) and healed faster (P = .006) than ACTH pigs. Thus, exogenous ACTH and restraint during gestation alters the HPA axis of the sow's offspring, and during stressful situations later in life health, and therefore welfare, may be compromised.

Behavioral and Hormonal Effects of Centrally Injected “Anxiogenic” Neuropeptides in Growing Pigs

Records of behavior (alertness, posture, oro-nasal responses, activity level, and vocalization pattern) were made in prepubertal pigs ( n = 6) during a 60-min period following central injections of equimolar (21 nmol) doses of porcine CRH (pCRH), urocortin (UCN), octadecaneuropeptide (ODN), or saline vehicle (SAL). Blood samples were also collected at 15-min intervals before, during, and after the test, and used to determine plasma cortisol, prolactin, and growth hormone concentrations. The pigs became excited and highly active after pCRH, and to a lesser extent following UCN administration, but were subdued when given ODN or SAL. None of the peptides significantly affected prolactin or growth hormone release, but both UCN, and especially pCRH, increased cortisol concentrations. The emotional responses induced by pCRH and UCN are consistent with observations in rodents, which indicate that centrally administered CRH-like peptides have anxiogenic effects. In contrast, ODN, which inhibits benzodiazepine binding at the GABA A receptor and is anxiogenic in rodents, lowered plasma cortisol and had no overt behavioral effects. Hence, at the dose administered, there was no evidence to indicate that ODN acted as an anxiogen in this species.

Evaluation of a low-dose synthetic adrenocorticotropic hormone stimulation test in clinically normal dogs and dogs with naturally developing hyperadrenocorticism

Objective To determine whether low doses of synthetic ACTH could induce a maximal cortisol response in clinically normal dogs and to compare a low-dose ACTH stimulation protocol to a standard high-dose ACTH stimulation protocol in dogs with hyperadreno-corticism. Design Cohort study. Animats 6 clinically normal dogs and 7 dogs with hyperadrenocorticism. Procedure Each clinically normal dog was given 1 of 3 doses of cosyntropin (1, 5, or 10 μg/kg [0.45, 2.3, or 4.5 μg/lb] of body weight, IV) in random order at 2-week intervals. Samples for determination of plasma cortisol and ACTH concentrations were obtained before and 30, 60, 90, and 120 minutes after ACTH administration. Each dog with hyperadrenocorticism was given 2 doses of cosyntropin (5 μg/kg or 250 μg/dog) in random order at 2-week intervals. In these dogs, samples for determination of plasma cortisol concentrations were obtained before and 60 minutes after ACTH administration. Results In the clinically normal dogs, peak cortisol concentration and area under the plasma cortisol response curve did not differ significantly among the 3 doses. However, mean plasma cortisol concentration in dogs given 1 μg/kg peaked at 60 minutes, whereas dogs given doses of 5 or 10 μg/kg had peak cortisol values at 90 minutes. In dogs with hyperadrenocorticism, significant differences were not detected between cortisol concentrations after administration of the low or high dose of cosyntropin. Ctinical Implications Administration of cosyntropin at a rate of 5 μg/kg resulted in maximal stimulation of the adrenal cortex in clinically normal dogs and dogs with hyperadrenocorticism. (J Am Vet Med Assoc 1999;214:1497-1501)

Determination of cortisol in plasma and urine by thin-layer chromatography and fluorescence derivatization with isonicotinic acid hydrazide

A simple, inexpensive and reliable method is described for the measurement of cortisol in guinea-pig plasma and urine by thin-layer chromatography (TLC) and densitometry. Plasma and urine were extracted with dichloromethane and the extracts chromatographed on aluminium-backed silica gel TLC plates with concentrating zones. The plates were then dipped into the isonicotinic acid hydrazide (INH) reagent (3 g INH, 5 g trichloroacetic acid, 300 mL ethanol). The fluorescence of the cortisol hydrazone was further increased by dipping the plates into chloroform-liquid paraffin (9:1,v/v). The fluorescence was determined densitometrically (excitation: 366 nm, cut-off filter: >460 nm). The fluorescence intensity was linearly dependent on the amount of cortisol between 1 ng and 200 ng; the coefficients of variation ranged between 6.3% (1 ng) and 1.4% (200 ng). The sensitivity of this method (<1 ng) enables the measurement of cortisol in the plasma or urine of saline-, ACTH- or cortisol-treated guinea-pigs. Comparison of the cortisol concentration measured by this method or by a TLC-radioimmunoassay technique gave correlation coefficients ofr=0.98 (plasma) andr=0.97 (urine). Thus, in conjunction with the isolation of cortisol by TLC, the densitometric measurement of the fluorescent hydrazone of cortisol is a sensitive and rapid routine means of determination of the concentration of cortisol in biological materials such as plasma or urine.

Effects of transport and indomethacin on telemetered body temperature and release of cortisol and prolactin in pre-pubertal pigs

Previous research indicates that acute physical stress (restraint) raises core temperature in growing pigs via a prostaglandin-dependent mechanism. This study investigated whether transport stress affects body temperature in pigs and whether any such changes might involve endogenous prostaglandins. Pre-pubertal boars (n = 7) were implanted with venous catheters and biotelemetry devices for the measurement of core temperature. They were transported for two hours, with and without indomethacin (IND) pretreatment, and blood samples were taken at 15 minute intervals for the determination of plasma cortisol and prolactin concentrations. The results indicated that, contrary to predictions, body temperature tended to fall during transport and that the effect was exaggerated by IND. By contrast, cortisol concentrations increased during transport and were unaffected by IND whereas the tendency for transport to stimulate prolactin release was reversed by IND.