Radioimmunoassay of cholecystokinin in human plasma

A sensitive radioimmunoassay for cholecystokinin (CCK) has been developed. Porcine CCK-33 was labelled by conjugation with 125I-hydroxyphenyl-propionic acid succinimide ester. Antibodies were raised against porcine CCK-33 covalently coupled to egg albumin. Plasma samples were extracted with 96% ethanol prior to assay. Free and bound hormone were separated by dextran-coated charcoal. The antibodies bound CCK-8 and CCK-33 with equimolar potency. The assay detection limit was 1 pmol/l plasma. Within and between assay coefficients of variation were +/- 12.7 and 13.0% at mean plasma CCK concentrations of 13.2 and 13.6 pmol/l. The concentration of CCK in 47 normal fasting subjects ranged from undetectable to 22 pmol/l. Ingestion of a mixed meal in 9 normal subjects increased the plasma concentration from 8.3 +/- 2.5 S.E. to 24.4 +/- 6.5 pmol/l.     

Radioimmunoassay of bromperidol and haloperidol in human and canine plasma

In order to check the specificity of a radioimmunological assay for haloperidol and bromperidol reported by Michiels et al. (1) and commercialized by IRE (Fleurus, Belgium), the assay was performed on canine and human plasma samples with and without extraction. After a single dose of bromperidol in dogs, plasma levels obtained with and without extraction were similar. In man the plasma levels for bromperidol after a single administration were markedly lower after extraction. The same was true for haloperidol plasma levels after chronic dosing in man. The findings suggest that the direct radioimmunoassay of haloperidol or bromperidol with the commercialized kit, lacks specificity of humans, possibly because of the presence of one or more polar metabolites.     

Radioimmunoassay of 8-arginine-vasopressin (antidiuretic hormone) in human plasma

A radioimmunoassay for 8-arginine-vasopressin (AVP) measurement in human plasma has been developed and evaluated, using a commercial preparation of an antibody of AVP. Detection limit of the assay was 0.4 pg. A simple acetone extraction procedure gave a recovery of 65% of added [125I]AVP. The overall sensitivity in the assay was 1.0 pg/ml when 2 ml plasma samples were extracted. The antigenic sites of the employed antibody seemed to be a combination of amino acid residues in the tripeptide tail and the pentapeptide ring. This can explain that the antibody was almost completely insensitive to chemically or enzymatically degraded AVP. The inter-assay coefficient of variation for the control plasma pools averaged 17%. A good correlation to plasma osmolalities above 290 has been found. AVP level in recumbent subjects (n = 8) with plasma osmolalities in the normal range was 2.8 +/- 1.0 pg/ml (mean +/- SD) and in ambulatory subjects (n = 10) on ad lib. water intake 4.5 +/- 1.9 pg/ml (mean /+- SD).     

Characterization of immunoreactive dynorphin B and beta-endorphin in human plasma

Dynorphins and beta-endorphin in human plasma were characterized and studied quantitatively using radioimmunoassay, high-performance liquid chromatography (HPLC), and mass spectrometry. Most immunoreactive (ir) dynorphin B and beta-endorphin in human plasma coeluted with authentic peptides in analysis. Dynorphin A was not detected. Added to human plasma it was rapidly converted into Leu-enkephalin-Arg6 followed by elimination of the C-terminal arginine after prolonged incubation. The rate of dynorphin A conversion was estimated at 40 pmol/min/microl plasma. This process was inhibited by the thiol protease inhibitor, PHMB and by EDTA. Dynorphin B, alpha-neoendorphin and big dynorphin were virtually not metabolized by plasma proteases under the same conditions. beta-endorphin was processed into beta-endorphin(1-19) and the corresponding C-terminal counterpart beta-endorphin(20-31) at a rate of about 25 pmol/min/microl of plasma. Based on the above data, a reliable strategy was established to measure dynorphin B- and beta-endorphin-ir in human plasma samples. The basal levels in a male control group were 0.99 +/- 0.11 (n = 11) and 16.3 +/- 1.5 (n = 11) fmol/ml plasma, respectively.     

Further studies on irreversible inhibition of dopamine-beta-hydroxylase by cysteine

In the process of studying the inhibition of dopamine-beta-hydroxylase by cysteine, we observed an interesting relationship between cysteine and catalase. This suggests that two different patterns of the inhibition of dopamine-beta-hydroxylase exist; the one is reversible inhibition, the other is irreversible inhibition. In the present experiments, we have particularly investigated the irreversible inhibition of dopamine-beta-hydroxylase by cysteine, and the following conclusions were drawn: (a) The rate of the inhibitory effect of cysteine pretreatment on dopamine-beta-hydroxylase is independent of a function of the temperature, while at temperature above 20 degrees C the extent of the reversibility of cysteine inhibition by N-ethylmaleimide is temperature dependent. (b) The rate of inhibitory effect is dependent on the time of cysteine pretreatment at 37 degrees C, and the ability of N-ethylmaleimide to reverse the cystine-induced inhibition is gradually diminished by increasing the time of cysteine pretreatment. (c) The inhibitory effect of cysteine pretreatment (37 degrees C, 10 min) is not reversed by the addition of Cu++. (d) In the presence of higher concentrations of catalase, the cystein-induced inhibition is recovered after the addition of equimolar concentration of N-ethylmaleimide. (e) Both inhibitions of cysteine on dopamine-beta-hydroxylase with and without pretreatment were the noncompetitive type to the substrate, tyramine.     

Synaptic innervation of enkephalinergic neurons by axon terminals immunoreactive to dopamine-beta-hydroxylase in the rat area postrema

A preembedding double immunostaining technique was used to study synaptic relations between enkephalin-like immunoreactive and dopamine-beta-hydroxylase-like immunoreactive neurons in the rat area postrema. Enkephalin-like immunoreactive neuronal perikarya and dendrites were found to receive synapses from dopamine-beta-hydroxylase-like immunoreactive axon terminals. Synapses were also found between the same dopamine-beta-hydroxylase-like immunoreactive neurons. Compared with our previous study, the present results provide morphological evidence that dopaminergic and noradrenergic neurons have different synaptic relations with enkephalinergic neurons, suggesting that physiological functions, especially those related to enkephalinergic neurons, may be different from each other in the area postrema.     

Comparative study of plasma dopamine-beta-hydroxylase activities in noradrenaline-secreting and adrenaline-secreting phaeochromocytomae

1. Circulating dopamine-beta-hydroxylase activities were measured in two hypertensive patients, with a phaeochromocytoma tumour. Tumours were found to differ by their secretory properties, one secreting noradrenaline, the other adrenaline. After removal of the tumour, the plasma dopamine-beta-hydroxylase activities in both patients gradually decreased to reach a stable value in correlation with urinary, excreted catecholamine levels. The only difference was the rate of the plasma enzyme activity decrease. 2. Thus, it appeared that some phaeochromocytomae are able to secrete dopamine-beta-hydroxylase in addition to catecholamines. Therefore, dopamine-beta-hydroxylase activity measurements may be of interest: (1) in determining secretory properties of phaeochromocytoma tumours, and (2) in following the evolution during the postoperative period.     

Radioimmunoassay of bound and free melatonin in plasma

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.     

Radioimmunoassay of glutathione peroxidase in human serum

Glutathione peroxidase (GSHPx) was purified from human serum and used for immunization of rabbits. Antiserum bound up to 75% of added 125I-GSHPx after precipitation with a second antibody. Human serum, but not sera from eight animal species, inhibited the binding of labelled GSHPx, indicating that the antiserum did not react with GSHPx from these species. GSHPx could be measured in less than 10 microliters of human serum by radioimmunoassay. In sera with widely varying selenium concentrations (0.1-2.9 mumol/l) the amount of GSHPx protein (0.3-6.3 mg/l) was strongly correlated with GSHPx activity (r = 0.94) and it was also correlated with serum selenium concentrations (r = 0.64). This indicates that GSHPx protein may be a valuable biological marker of selenium status. In samples with serum selenium concentrations of 0.8-1.2 mumol/l, the concentration of GSHPx was 3.3 (0.4) mg/l (mean (S.D.)), or 0.04 (0.005) mumol/l. This corresponded to 0.16 (0.02) mumol/l of GSHPx selenium and 16% (2.8)% of total serum selenium. The data suggest that the method can be used to measure the proportion of serum selenium that is located in GSHPx. Following storage of serum at room temperature, both serum GSHPx protein and activity declined, but addition of glutathione protected both GSHPx protein and activity.     

Radioimmunoassay of thyrotropin releasing hormone in plasma and urine

A sensitive and specific radioimmunoassay has been developed capable of measuring thyrotropin releasing hormone (TRH) in extracted human plasma and urine. All of three TRH analogues tested had little cross-reactivity to antibody. Luteinizing hormone releasing hormone, lysine vasopressin, rat growth hormone and bovine albumin were without effect, but rat hypothalamic extract produced a displacement curve which was parallel to that obtained with the synthetic TRH. Sensitivity of the radioimmunoassay was 4 pg per tube with intraassay coefficient of variation of 6.2-9.7%. Synthetic TRH could be quantitatively extracted by methanol when added to human plasma in concentration of 25, 50 and 100 pg/ml. TRH immunoreactivity was rapidly reduced in plasma at 20 degrees C than at 0 degrees C, but addition of peptidase inhibitors, FOY-007 and BAL, prevented the inactivation of TRH for 3 hr at 0 degrees C. The TRH in urine was more stable at 0 degrees C than 20 degrees C, and recovered 75 +/- 4.6% hr after being added. The plasma levels of TRH were 19 pg/ml or less in normal adults and no sex difference was observed. The rate of disappearance of TRH administered i.v. from the blood could be represented as half-times of 4-12 min. Between 5.3-12.3% of the injected dose was excreted into urine within 1 hr as an immunoreactive TRH. These results indicate the usefulness of TRH radioimmunoassay for clinical investigation.     

Radioimmunoassay of free genistein in human serum

Two radioimmunoassay (RIA) systems for genistein have been established, based on polyclonal antibodies against genistein-4'-O-(carboxymethyl)ether-bovine serum albumin and genistein-7-O-(carboxymethyl)ether-bovine serum albumin conjugates. The sensitivities of assays were 4.44 and 10.4 fmol (1.2 and 2.8 pg)/tube, respectively, the intraassay coefficients of variation ranged from 3.54 to 9.30%, the interassay C.V. varied from 6.72 to 19.7%, depending on the type of method and on genistein concentration. The cross-reactivities with other chemically related compounds (with exception of genistein derivatives at the position used for construction of the immunogen) were 5.5 and 6.1% for daidzein and 3.9 and 0.04% for formononetin in RIAs using reagents prepared through positions 4'- and 7- of genistein, respectively. The method was used for measurement of genistein levels in 26 omnivore subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The values obtained in ether extracts from human sera were almost identical for both RIA systems, indicating that both RIAs measure the same entity.     

Radioimmunoassay of human Hageman factor (factor XII)

A secific, sensitive, and reproducible radioimmunoassay for human Hageman factor (HF, factory XII) has been developed with purified human HF and monospecific rabbit antibody. Precise measurements of HF antigen were possible for concentrations as low as 0.1% of that in normal pooled plasma. A good correlation (correlation co-efficient = 0.82) existed between the titers of HF measured by clot-promoting assays and radioimmunoassays among 42 normal adults. Confirming earlier studies, HF antigen was absent in Hageman trait plasma, but other congenital deficient plasmas, including those of individuals with Fletcher trait and Fitzgerald trait, contained normal amounts of HF antigen. HF antigen was reduced in the plasmas of patients with disseminated intravascular coagulation or advanced liver cirrhosis, but it was normal in those of patients with chronic renal failure or patients under treatment with warfarin. HF antigen was detected by this assay in plasmas of primates, but not detectable in plasmas of 11 nonprimate mammalian and one avian species.     

Role of the kidney in regulating plasma immunoreactive beta-melanocyte-stimulating hormone

An analysis of the factors that influence the increase in plasma immunoreactive beta-melanocyte-stimulating hormone (beta-MSH) concentration in chronic renal failure showed that: (a) the increase correlated with the increase in serum creatinine concentrations; (b) beta-MSH was not cleared from the plasma by haemodialysis; (c) beta-MSH concentrations increased with length of time on dialysis and increased further after bilateral nephrectomy but there was no further increase with time; (d) beta-MSH levels decreased to normal after renal transplantation; and (e) beta-MSH was excreted in urine only when plasma levels rose to well above those of chronic renal failure (in Nelson's syndrome). These findings suggest that the kidney regulated plasma beta-MSH by a non-excretory mechanism and is the major site of beta-MSH metabolism.     

Enantioselective analysis of N-hydroxymexiletine glucuronide in human plasma for pharmacokinetic studies

CO2 reactivity of cerebral hemoglobin concentration was studied in 16 healthy term neonates on days 1 and 4 after birth using the near infrared spectrophotometry (NIRS) technique. The aim was to establish data on the physiological range of CO2 reactivity in healthy newborns and to investigate the influence of postnatal age on it. The CO2 reactivity measured by NIRS is expressed as the change of the total cerebral hemoglobin concentration (tHbR) per change of CO2 tension in micromol/l/kPa. We evaluated CO2 reactivity during increases and decreases of transcutaneous CO2 partial pressure and found in our methodological setting the data of the increases more reliable. In all infants but 1 we found a tHbR on day 1 with a mean value of 8.19 micromol/l/kPa (-1.39 to 18.87), in all infants on day 4 with a mean value of 9.54 micromol/l/kPa (2.76-25. 88). There is a trend to higher values between day 1 and day 4 (difference = 2.25 micromol/l/kPa; p = 0.08). The noninvasive NIRS technique enabled us to test the cerebrovascular CO2 reactivity of the tHbR for the first time in healthy term newborns. Data on its physiologic range and variability are presented and compared to findings from ventilated infants and other age groups. As the CO2 reactivity might be an indicator for infants at risk of cerebral damage, it is necessary to have data on the physiological range of this parameter.     

Noradrenergic innervation of the hypothalamus of rhesus monkeys: distribution of dopamine-beta-hydroxylase immunoreactive fibers and quantitative analysis of varicosities in the paraventricular nucleus

The distribution of noradrenergic processes within the hypothalamus of rhesus monkeys (Macaca mulatta) was examined by immunohistochemistry with an antibody against dopamine-beta-hydroxylase. The results revealed that the pattern of dopamine-beta-hydroxylase immunoreactivity varied systematically throughout the rhesus monkey hypothalamus. Extremely high densities of dopamine-beta-hydroxylase-immunoreactive processes were observed in the paraventricular and supraoptic nuclei, while relatively lower levels were found in the arcuate and dorsomedial nuclei and in the medial preoptic, perifornical, and suprachiasmatic areas. Moderate levels of dopamine-beta-hydroxylase immunoreactivity were found throughout the lateral hypothalamic area and in the internal lamina of the median eminence. Very few immunoreactive processes were found in the ventromedial nucleus or in the mammillary complex. Other midline diencephalic structures were found to have high densities of dopamine-beta-hydroxylase immunoreactivity, including the paraventricular nucleus of the thalamus and a discrete subregion of nucleus reuniens, the magnocellular subfascicular nucleus. A moderate density of dopamine-beta-hydroxylase immunoreactive processes were found in the rhomboid nucleus and zona incerta whereas little dopamine-beta-hydroxylase immunoreactivity was found in the fields of Forel, nucleus reuniens, or subthalamic nucleus. The differential distribution of dopamine-beta-hydroxylase-immunoreactive processes may reflect a potential role of norepinephrine as a regulator of a variety of functions associated with the nuclei that are most heavily innervated, e.g., neuroendocrine release from the paraventricular and supraoptic nuclei, and gonadotropin release from the medial preoptic area and mediobasal hypothalamus. Additionally, quantitative analysis of dopamine-beta-hydroxylase-immunoreactive varicosities was performed on a laser scanning microscope in both magnocellular and parvicellular regions of the paraventricular nucleus of the hypothalamus. The methodology employed in this study allowed for the high resolution of immunoreactive profiles through the volume of tissue being analyzed, and was more accurate than conventional light microscopy in terms of varicosity quantification. Quantitatively, a significant difference in the density of dopamine-beta-hydroxylase-immunoreactive varicosities was found between magnocellular and parvicellular regions, suggesting that parvicellular neurons received a denser noradrenergic input. These differential patterns may reflect an important functional role for norepinephrine in the regulation of anterior pituitary secretion through the hypothalamic-pituitary-adrenal stress axis.     

Toward psychological studies of human freedom.

Outlines central positions and controversies concerning liberty and freedom as represented in social and political philosophy. Distinctions of importance to psychologists such as those among negative liberty, positive liberty, opportunities, resources, and accomplishment of acts, are discussed. Psychological concepts and data arising from studies of attribution, reactance, dissonance, personal causation, and psychotherapy are examined. The important distinction between freedom as defined by external criteria and freedom as personal experience is explored in conjunction with the problem of freedom as reality vs freedom as illusion. It is proposed that direct inquiry of articulate respondents concerning the experience of freedom and the conditions of its occurrence is a meaningful way to explore the phenomenon of freedom within a deterministic framework. Data from interviews with 12 20–40 yr old undergraduates or colleagues of the author are presented with suggestions for further research. (French summary) (36 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Validated high-performance liquid chromatographic assay for the determination of promazine in human plasma. Application to pharmacokinetic studies

A high-performance liquid chromatographic method for the determination of promazine in human plasma is described. The assay involves a single-step liquid-liquid extraction using pentane-2-propanol (98:2, v/v). The analyte of interest and the internal standard chlorpromazine were separated on a Spherisorb CN column using a mobile phase of acetonitrile-50 mM ammonium acetate (9:1, v/v). Electrochemical detection was achieved using an applied potential of +750 mV. The assay was validated according to international requirements prior to application to a pharmacokinetic study and was found to be specific, accurate and precise with a linear range of 0.25-25 ng ml(-1).