Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry

Gas chromatography mass spectrometry with selected ion monitoring has been used to develop a method for the quantification of ketotifen and its demethylated, 10-hydroxy and 10-hydroxy demethylated metabolites in human plasma. The minimum detectable concentrations for ketotifen and its demethylated metabolites were 50 pg ml-1 and 300 pg ml-1 for the 10-hydroxy metabolite. The methodology has been applied in studies of the kinetics of the drug in man, and plasma levels of the unchanged drugs and its metabolites in free and conjugated form are reported.     

Plasma vasopressin, renin activity, and aldosterone responses to maximal exercise in active college females

The effect of maximal treadmill exercise on plasma concentrations of vasopressin (AVP); renin activity (PRA); and aldosterone (ALDO) was studied in nine female college basketball players before and after a 5-month basketball season. Pre-season plasma AVP increased (p less than 0.05) from a pre-exercise concentration of 3.8 +/- 0.5 to 15.8 +/- 4.8 pg X ml-1 following exercise. Post-season, the pre-exercise plasma AVP level averaged 1.5 +/- 0.5 pg X ml-1 and increased to 16.7 +/- 5.9 pg X ml-1 after the exercise test. PRA increased (p less than 0.05) from a pre-exercise value of 1.6 +/- 0.6 to 6.8 +/- 1.7 ngAI X ml-1 X hr-1 5 min after the end of exercise during the pre-season test. In the post-season, the pre-exercise PRA was comparable (2.4 +/- 0.6 ngAI X ml- X hr-1), as was the elevation found after maximal exercise (8.3 +/- 1.9 ngAI X ml- X hr-1). Pre-season plasma ALDO increased (p less than 0.05) from 102.9 +/- 30.8 pg X ml-1 in the pre-exercise period to 453.8 +/- 54.8 pg X ml-1 after the exercise test. In the post-season the values were 108.9 +/- 19.4 and 365.9 +/- 64.4 pg X ml-1, respectively. Thus, maximal exercise in females produced significant increases in plasma AVP, renin activity, and ALDO that are comparable to those reported previously for male subjects. Moreover, this response is remarkably reproducible as demonstrated by the results of the two tests performed 5 months apart.     

The monitoring of the menstrual status of female athletes by salivary steroid determination and ultrasonography

This study was designed to evaluate whether traditional plasma hormone determinations can be adequately replaced by measurements of salivary hormones. Eleven young sportswomen with menstrual irregularities attributed to strenuous physical exercise participated in this study. Mean body weight expressed as a percentage of ideal body weight was 92%, SD 4%. Their mean weekly training distance was 35 km, SD 15. Basal plasma endocrinological measurements revealed a hypo-oestrogenic status (mean plasma oestradiol values: 22 pg.ml-1, SD 8.8), and a deficient luteal phase (mean plasma progesterone: 2.9 ng.ml-1, SD 2.1). Pre-exercise salivary sex steroids were low. Salivary progesterone levels were 39.3 pg.ml-1, SD 9.5 (normal ranges in saliva: 25-60 pg.ml-1), salivary oestrone (E1) was 12.2 pg.ml-1, SD 2.3 (normal ranges in saliva: 7.5-25 pg.ml-1), and salivary oestradiol (E2) less than 1.9 pg.ml-1, SD 1.1 (normally 1.0-10.0 pg.ml-1). After a 21-km run, all salivary steroids appeared to increase. Mean salivary testosterone levels increased by 15.2% and salivary progesterone by 14.8%. Mean salivary oestrogens also increased (E1: +13.9%; E2: +21.1%). These findings confirm the results of earlier studies which found higher post-exercise plasma sex steroid levels. Since salivary measurements are believed to reflect non-protein-bound, thus free steroid levels, the results obtained by these techniques may provide a more realistic picture of the hormonal effects of physical exercise. In future, more accurate, cost-effective and easier techniques for salivary measurements may offer additional advantages.     

Bis(trifluoromethyl)aryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of low levels of clonidine in plasma

A gas chromatographic mass spectrometric assay for clonidine in plasma with a detection limit of a few picograms per ml was required. The p-trifluoromethylbenzyl, pentafluorobenzyl and pentafluorobenzoyl derivatives of clonidine were synthesized and the electron capture negative ion chemical ionization mass spectra of these compounds show extensive fragmentation with prominent ions at m/z 35 and 37 due to the two chlorine atoms in the clonidine molecule. Selected ion monitoring of specific high mass ions in these mass spectra indicated that the required sensitivity could not be obtained with these derivatives. Several bis(trifluoromethyl)pyrimidines were synthesized and these compounds were found to give an intense negative ion current under conditions of resonance electron capture. Consequently, a derivative of clonidine containing a bis(trifluoromethyl)aryl group was synthesized by reacting the drug with 3,5-bis(trifluoromethyl)benzoyl chloride. The negative ion mass spectrum of the reaction product has a base peak at m/z 673 and, when this ion is specifically monitored, an amount of derivative equivalent to 1 picogram of clonidine can be detected. This allowed the development of an assay for clonidine in plasma with a precision of 8% (SD) at 50 pg ml-1, 22% (SD) at 20 pg ml-1 and a lower limit for quantitative determination of 10 pg ml-1. Plasma concentrations of clonidine in 10 subjects given a single 50 micrograms oral dose are reported.     

Development of blood testosterone in the embryo and young chick after precocious bursectomy

The effect of bursa of Fabricius on the endocrine function of the chick testes was studied in vivo by comparing plasma testosterone levels from 48 h before hatch to 16 weeks of age in both intact and bursectomized chicken. Early bursectomy was performed at 80 h of incubation. Post surgery survival was low (12% at 1 week). In controls, plasma testosterone levels were found to be low (100-200 pg. ml-1) from 48 h before to 48 h after hatch, then to raise up to a plateau (2,200 pg. ml-1) at 6 weeks. After bursectomy, values were first higher than in intact (210-440 pg. ml-1 from 48 h before/after hatch and 515 vs 300 pg.ml-1 at 3 days) but no difference could be further detected from 1 to 16 weeks of age. It is suggested that, in addition to the effect of androgen on bursa of Fabricius, the later reciprocally influences the gonadotropic axis during the early stage of development.     

Plasma renin activity, aldosterone and catecholamine levels when swimming and running

The purpose of this study was to determine the response of plasma renin activity (PRA), plasma aldosterone concentration (PAC) and catecholamines to two graded exercises differing by posture. Seven male subjects (19-25 years) performed successively a running rest on a treadmill and a swimming test in a 50-m swimming pool. Each exercise was increased in severity in 5-min steps with intervals of 1 min. Oxygen consumption, heart rate and blood lactate, measured every 5 min, showed a similar progression in energy expenditure until exhaustion, but there was a shorter time to exhaustion in the last step of the running test. PRA, PAC and catecholamines were increased after both types of exercise. The PRA increase was higher after the running test (20.9 ng AngI X ml-1 X h-1) than after swimming (8.66 ng AngI X ml-1 X h-1). The PAC increase was slightly greater after running (123 pg X ml-1) than swimming (102 pg X ml-1), buth the difference was not significant. Plasma catecholamine was higher after the swimming test. These results suggest that the volume shift induced by the supine position and water pressure during swimming decreased the PRA response. The association after swimming compared to running of a decreased PRA and an enhanced catecholamine response rule out a strict dependence of renin release under the effect of plasma catecholamines and is evidence of the major role of neural pathways for renin secretion during physical exercise.     

The determination of terbutaline in human plasma by selected ion monitoring of the t-butyldimethylsilyl ether

A method is described for the quantitative determination of terbutaline in 2 ml human plasma. The drug is extracted from plasma as the terbutaline tetraphenylboron ion pair and determined by gas chromatography mass spectrometry of its t-butyldimethylsily ether. Salbutamol is used as internal standard. Quantification is achieved by selected ion monitoring of the ion m/z 482 derived from t-butyldimethylsilyl terbutaline and m/z 495 from t-butyldimethylsilyl salbutamol. The detection limit was estimated to be 250 pg terbutaline ml-1 plasma. The coefficient of variation at the level of 1 ng terbutaline ml-1 was 4.1% (n = 5).     

The role of posture on the changes in plasma atrial natriuretic factor and arginine vasopressin levels during immersion

In seven healthy male volunteers we investigated changes in plasma atrial natriuretic factor [( ANF]), arginine vasopressin [( AVP]) and plasma volume (PV) during supine immersion. Twenty minutes head-out water immersion in a supine position in a thermo-neutral water bath attenuated the increase in PV induced by 20 min in a supine position in air, but increased the mean plasma [ANF] from 32.0 pg.ml-1, SEM 5.1 to 53.3 pg.ml-1, SEM 3.6 and decreased the mean plasma [AVP] from 1.4 pg.ml-1, SEM 0.1 to 0.9 pg.ml-1, SEM 0.04. Simultaneously, diuresis and natriuresis increased markedly. During a 20-min control period in the supine posture without immersion, PV, plasma [ANF] and [AVP] remained unaffected while diuresis and natriuresis did not increase to the same extent. These data suggest that an increase in the central blood volume induced by a weak external hydrostatic pressure during supine immersion triggered the changes in plasma [ANF] and [AVP] and that the increase was probably due to a shift of blood volume from peripheral to central vessels. The changes in plasma [ANF] contributed to the changes in natriuresis.     

Evidence from plasma progesterone concentrations for male-induced ovulation in the brush-tailed bettong, Bettongia penicillata.

Female brush-tailed bettongs, Bettongia penicillata, were housed with either an intact or vasectomized male or isolated from males in the peripartum period. Development of the quiescent corpus luteum formed at the post partum oestrus was initiated by removing the pouch young. Blood samples for analysis of plasma progesterone were collected from the females 2 days before removal of pouch young, daily for 5 or 6 days and then 2-3 times each week until 19 days after removal of pouch young. Plasma progesterone profiles were similar in pregnant and nonpregnant cycles. There was an early progesterone peak (1206 +/- 121 pg ml-1, mean +/- SEM; n = 16) between days 2 and 5 after removal of pouch young, and a second period of high concentrations (greater than 800 pg ml-1) before birth on day 17.4 +/- 0.2 (n = 16). The interval between the early peak and birth was 14 or 15 days. On five of 34 occasions, no increases in plasma progesterone concentrations occurred after removal of pouch young. On 12 of 15 occasions for 13 females that had been isolated from males post partum, plasma progesterone concentrations also remained low (less than 100 pg ml-1) and did not change after removal of pouch young. Females that showed no increases in plasma progesterone concentration after removal of pouch young had significantly lower (P less than 0.001) plasma progesterone concentrations while lactating than those females that did undergo a cycle after removal of pouch young (60 +/- 4 pg ml-1, n = 17 and 225 +/- 23 pg ml-1, n = 30, respectively). Females isolated from males post partum, and monitored until day 12 after removal of the pouch young, and that showed no increases in progesterone in this period, had ovaries that contained no corpus luteum, only corpora albicantia and numerous atretic or developing follicles. We conclude that brush-tailed bettongs are induced ovulators, a characteristic described for only one other marsupial, Monodelphis domestica, from South America.     

Increased glucagon secretion during hyperthermia in a sauna

Plasma glucagon, adrenaline, noradrenaline, insulin and glucose concentrations were measured in 7 healthy young males during hyperthermia in a sauna bath: plasma glucagon levels increased from baseline values of 127.0 +/- 12.9 (SEM) pg X ml-1 to a maximum of 173.6 +/- 16.1 (SEM) pg X ml-1 at the 20th min of exposure. No change in plasma insulin and a slight increase in plasma glucose concentration were seen. Since a concomitant moderate increase in plasma catecholamine levels was also present, the adrenergic stimulus is believed to trigger glucagon release during hyperthermia. Diminished visceral blood flow, known to occur in sauna baths, may cause a decrease in the degradation of plasma glucagon and thus contribute to the elevated plasma glucagon levels.     

Quantification of plasma phenylethylamine by combined gas chromatography/electron capture negative ion mass spectrometry of the N-acetyl-N-pentafluorobenzoyl derivative

An ultrasensitive method capable of detection and quantification of beta-phenylethylamine in 1 ml of human plasma has been developed using gas chromatography/electron capture negative ion mass spectrometry. Phenylethylamine and tetra-deutero phenylethylamine internal standard in plasma were acetylated, extracted into organic solvent and then further acylated with pentafluorobenzoyl chloride. The N-acetyl-N-pentafluorobenzoyl-phenylethylamines were detected by high-resolution single ion monitoring of the molecular ions. Normal plasma levels were found to be 41.5 +/- 10.7 pg ml-1, in accordance with results of a previous high-performance liquid chromatographic method.     

A flow immunoassay for studies of human exposure and toxicity in biological samples

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.     

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Measurement of ergotamine in human plasma by triple sector quadrupole mass spectrometry with negative ion chemical ionization

By use of negative ion chemical ionization and collision-activated decomposition in a triple quadrupole mass spectrometer a method has been developed for the quantification of ergotamine in human plasma at levels down to 2 pg ml-1.     

Effects of physical exercise and anti-G suit inflation on atrial natriuretic factor plasma level

The purpose of this study was to measure the effect of enhanced venous return on atrial natriuretic factor (ANF) secretion during exercise and upright posture and the consequences on renin angiotensin aldosterone system (RAAS) activity. Six healthy male subjects were submitted to four different procedures. All procedures were performed in the same position, i.e. riding on a support with legs hanging. Two procedures were performed at rest: the subjects were studied after a 25-min rest in this position, with and without the lower limb fitted with an anti-G suit inflated to 60 mmHg. Two procedures were carried out with physical exercise; arm-cranking was performed in the same position with and without the anti-G suit inflated to 60 mmHg. Venous blood was collected before and after each procedure in order to measure plasma ANF, plasma aldosterone concentration (PAC), plasma renin activity (PRA), corticotrophin (ACTH) and catecholamine level. The data mean +/- SEM showed that the ANF plasma level decreased significantly (p less than 0.05) from 32.5 +/- 4 to 28 +/- 6 pg.ml-1 after a 20-min rest in the upright posture, whereas this effect was absolished with anti-G suit inflation. Physical exercise with and without the anti-G suit increased the ANF level above control values (60 +/- 13.6 pg.ml-1 and 53 +/- 13 pg.ml-1): anti-G suit inflation had no significant effect. PRA increased after rest in an upright posture and during physical exercise; anti-G suit inflation abolished this increase in both conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. II. Endocrine and metabolic functions.

The present study examines the effect of subcutaneous pancreatic tissue grafts (SPTG) on endocrine and metabolic functions in streptozotocin (STZ)-induced diabetic rats using radioimmunoassay and biochemical techniques. SPTG survived even after 15 weeks of transplantation and significantly improved the weight of STZ-diabetic rats over a 15-week period. Although blood glucose-, cholesterol-, and glycosylated-haemoglobin (GHb) levels were not significantly lower in STZ-diabetic rats treated with SPTG, the values of these biochemical parameters were lower than those in untreated diabetic rats. Plasma and pancreatic immunoreactive C-peptide (IRCP) levels did not improve after SPTG (IRCP expressed as mean +/- standard deviation were 0.22 +/- 0.07, 0.072 +/- 0.02 and 0.08 +/- 0.03 pg ml-1 in the plasma non-diabetic diabetic and treated rats respectively, while IRCP levels in the pancreas of the non-diabetic, diabetic and treated rats were 433.8 +/- 0.1, 22.9 +/- 0.01 and 10.4 +/- 0.01 pg mg tissue-1 respectively). SPTG, however, improved plasma immunoreactive insulin (IRI) levels in both plasma and pancreas. IRI values in plasma were 54.7 +/- 13.6, 18.0 +/- 5.0 and 22.1 +/- 4.3 microUI ml-1 in non-diabetic, diabetic and treated rats respectively and were 277.3 +/- 37.1, 14.7 +/- 1.8 and 30.3 +/- 15.9 microIU micrograms tissue-1 in the pancreas of non-diabetic, diabetic and treated rats respectively. There was improvement in immunoreactive glucagon (IRG) levels after SPTG. IRG values in the plasma of non-diabetic, diabetic and treated rats were 147.0 +/- 10.7, 408.0 +/- 76.5 and 247.7 +/- 3 pg ml-1 respectively whereas, IRG measured in the pancreas was 1642.25 +/- 424.23, 1899.0 +/- 290.4 and 1714.1 +/- 301.98 pg micrograms tissue-1 in non-diabetic, diabetic and treated rats, respectively. The pancreas:plasma ratio of pancreatic hormones was deranged in untreated diabetes but improved after SPTG. In conclusion, SPTG significantly improved the weight gain, pancreatic insulin content, plasma IRG and pancreas: plasma ratio of IRCP, IRI and IRG. It also reduced blood glucose-, cholesterol-, and glycosylated-hemoglobin levels in STZ-diabetic rats.     

Effect of 1-desamino-8-D-arginine vasopressin (DDAVP) on vasopressin release and blood pressure during hemorrhage.

Whether or not 1-desamino-8-D-arginine-vasopressin (DDAVP) reduces blood pressure or affects the release of arginine vasopressin (AVP) and renin is controversial, although evidence suggests AVP and renin are important in maintaining blood pressure during hemorrhage. We therefore investigated the effect of DDAVP on endogenous release of AVP and renin and on blood pressure during hemorrhage in dogs. In the control group the hemorrhage was performed at a rate of 0.4 ml.kg-1.min-1 for 40 min from the femoral artery. The plasma AVP concentration and renin activity (PRA) increased progressively in response to the hemorrhage, from 7.5 +/- 0.5 to 40.3 +/- 7.3 pg.ml-1, and from 11.8 +/- 1.5 to 20.5 +/- 4.2 ng.ml-1.h-1, respectively, while blood pressure decreased slightly. In the DDAVP group, intravenous infusion of DDAVP (2.5 ng.kg-1.min-1 for 40 min) and hemorrhage were simultaneously performed. The plasma DDAVP concentration increased progressively to 218 +/- 21.0 pg.ml-1. There was no significant difference, however, between the control and DDAVP groups in the response of AVP, PRA and blood pressure. The results suggested that DDAVP may not affect the release of AVP and renin or blood pressure during hemorrhage.     

The influence of carotid sinus pressure on plasma atrial natriuretic peptide in anaesthetized rabbits

The effect of changes in carotid sinus perfusion pressure on plasma immunoreactive atrial natriuretic peptide (IR-ANP) was examined in anaesthetized rabbits, and the role of arterial pressure in mediating the changes in IR-ANP was assessed. Plasma IR-ANP was significantly greater (101.7 +/- 24.3 pg ml-1) when carotid sinus pressure was 60 mmHg than when it was 160 mmHg (27.1 +/- 8.6 pg ml-1). Mean arterial pressure (MAP) was significantly greater when carotid sinus pressure was controlled at 60 mmHg compared to when it was 160 mmHg, but right atrial pressure (RAP) was not significantly different at the two carotid sinus pressures. The administration of hexamethonium attenuated the changes in MAP and heart rate (HR) which occurred in response to alterations in carotid sinus pressure, and abolished the change in plasma IR-ANP. The results suggest that an inverse relationship exists between carotid sinus pressure and plasma IR-ANP, and that the release of ANP in response to a reduction of carotid sinus pressure is mediated by the associated haemodynamic changes.     

Central effects of atrial natriuretic peptide on plasma catecholamines, vasopressin, renin and beta-endorphin and on renal excretory functions in the dog

Atrial natriuretic peptide (ANP) has been identified in the central nervous system and its participation in regulation of various regulatory brain functions has been postulated. To elucidate whether central ANP influences endocrine systems related to blood pressure regulation and renal excretory functions, effects of infusion of ANP at a rate of 120 ng.min-1 into the third cerebral ventricle on plasma level of epinephrine (E), norepinephrine (NE), renin, vasopressin and beta-endorphin as well as on excretion of urine, sodium, potassium (UKV) solutes and free water (CH2O) were investigated in conscious dogs. Significant decrease of plasma E from 77.6 +/- 7.0 to 62.1 +/- 4.8 pg.ml-1 and of NE from 345.5 +/- 20.7 to 286.4 +/- 15.0 pg.ml-1 was found at the end of 30 min lasting ANP infusion. Significant elevation of PRA and UKV and a decrease in CH2O were found 60 min after ANP infusion. No significant changes in other variables were found. In time control experiments plasma hormones concentration and renal excretory functions were not significantly influenced. The results suggest that central ANP may affect the sympatho-adrenal outflow.