Enzyme immunoassay for arginine vasopressin (AVP). 2. Serum interference and the measurement of plasma samples

The effect of serum interference on enzyme immunoassay (EIA) for arginine vasopressin (AVP) was studied. Before measurement, plasma was applied on an affinity chromatography to remove AVP and then treated with the acetone-ether method which is usually used for AVP extraction of plasma samples. The AVP-free plasma extract was used to obtain a plasma AVP standard which was compared with various buffer standards of AVP. As a result, the enzyme activities of plasma AVP standard were always lower than those of buffer standards even though the concentration of buffer was increased or added by gelatin. Therefore, EIA for AVP was clinically applicable by using the AVP-free plasma for the standard. Plasma samples were drawn in various clinical conditions and measured for AVP by plasma AVP standard to examine reproducibility and correlation with radioimmunoassay (RIA). The values measured repetitively for the same samples were reproducible. The values measured by EIA correlated well with those measured by RIA (y = 0.67 + 1.13x, r = 0.96). These indicates that EIA for AVP could be applied clinically to measure physiological levels of plasma AVP.     

HPLC-monitoring of AZT in HIV-infected patient's plasma: a critical study.

3'-azido-2', 3'-deoxythymidine (AZT) concentrations in spiked human plasma were determined by means of reversed-phase high performance liquid chromatography (RP-HPLC). Samples were first cleaned-up for analysis using solid-phase extraction (SPE) columns filled with Silipore C18. In the concentration range comprising usual peak plasma concentrations during AZT therapy (0.1-20 mumol/l, i.e. 0.026-5.34 micrograms/ml) mean efficiency of the extraction procedure reached as high as 75.3% of original AZT concentrations in standard unextracted aqueous solutions. Replicate analyses in this range gave satisfactory intra-assay precision and reproducibility with coefficient of variation less than 11.3%. Calibration curves both in water and plasma showed good linearity (r > 0.999). The detection limit in plasma was 2 mumol/l, i.e. 5.3 ng per a 20 microliter of sample injected to the HPLC column. Plasma levels of AZT after a single dose administration, determined by HPLC and RIA showed rather poor correlation (r = 0.8900). In RIA about 1.7-4.5 times higher concentration values were obtained in a relatively short time, and, consequently, this method may better fulfil the needs of routine drug monitoring.     

Detection of nevirapine in plasma using thin-layer chromatography

BACKGROUND: Nevirapine (NVP) is widely prescribed in resource-poor settings to pregnant women for treatment and prevention of HIV infection. High rates of misreported adherence, however, have compelled clinicians to find alternative methods to ensure systemic drug exposure. This report describes a fast, inexpensive thin-layer chromatography (TLC) method to detect the presence of NVP in human plasma. METHODS: Human plasma was spiked with various concentrations of NVP. NVP was subsequently isolated using solid-phase extraction and visualized with TLC. Clinical samples with NVP concentrations predetermined by high-performance liquid chromatography were used to validate the TLC method. RESULTS: NVP was detected at concentrations as low as 60 ng/mL. The lower limit of detection was set at 100 ng/mL due to the clear spot definition at this concentration. The turnaround time for assay results averages several hours, and costs associated with the assay are considerably below standard drug quantitation techniques. CONCLUSION: TLC provides a rapid, sensitive, and economical tool to qualitatively measure NVP in plasma. This method offers clinicians in resource-poor settings an alternative approach for measuring adherence, particularly in developing-world regions where NVP use is common and there is an immediate need to prevent mother-to-child HIV transmission.     

Development of a solid-phase radioimmunoassay for the determination of arginine-vasopressin in urine

A new solid-phase radioimmunoassay (RIA) has been developed for measuring arginine-vasopressin (AVP) in urine. AVP is first extracted from urine by adsorption on Vycor glass powder and eluted with acetone-water (60:40). The mean recovery is 75.3 +/- 2.2% (n = 18). The organic extract is evaporated to dryness and reconstituted in the assay buffer. Aliquots of this extract are then incubated with 125I-AVP in polystyrene LKB tubes previously coated with the antiserum (1:50000) for 48 hours. The free radioactive fraction is removed by aspiration and the tubes are counted. Values correlate well with those obtained by liquid-phase RIA using dextran-charcoal. Urinary AVP concentrations in normal Sprague-Dawley rats and rats with varying degrees of hydration have been measured.     

Development of a Solid-Phase Radioimmunoassay for the Determination of Arginine-Vasopressin in Urine

A new solid-phase radioimmunoassay (RIA) has been developed for measuring arginine-vasopressin (AVP) in urine. AVP is first extracted from urine by adsorption on Vycor glass powder and eluted with acetone-water (60:40). The mean recovery is 75.3 ± 2.2% (n =18). The organic extract is evaporated to dryness and reconstituted in the assay buffer. Aliquots of this extract are then incubated with ¹²⁵I-AVP in polystyrene LKB tubes previously coated with the antiserum (1:50000) for 48 hours. The free radioactive fraction is removed by aspiration and the tubes are counted. Values correlate well with those obtained by liquid-phase RIA using dextran-charcoal. Urinary AVP concentrations in normal Sprague-Dawley rats and rats with varying degrees of hydration have been measured.     

Development of a radioimmunoassay for human metallothionein

We report the development of a radioimmunoassay (RIA) for human metallothionein (MT). The MT was isolated from the liver of a patient with primary biliary cirrhosis and used to raise high titer antibodies in rabbits. The RIA was developed using Bolton-Hunter labeled MT as label and purified MT as standard. The detection limit of the assay was 0.4 ng/ml and concentrations between 0.8 and 100 ng/ml could be routinely measured. Dilutions of plasma samples containing high endogenous MT concentrations and tissue extracts in assay buffer showed parallelism with the standard curve and MT added to plasma could be fully recovered.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25-100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25-100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Development and validation of a sensitive radioimmunoassay for progesterone estimation in unextracted mithun (Bos frontalis) plasma

The objective of the study was to develop and validate a simple, reliable, and highly sensitive radioimmunoassay (RIA) for progesterone determination in mithun (Bos frontalis) plasma. The RIA was carried out in 20 microL unextracted mithun plasma. The progesterone standards ranging from 2 to 500pg/20 microL/tube were prepared in charcoal-treated hormone-free plasma. The sensitivity of the RIA procedure was 2 pg progesterone/20 microL/tube, which corresponds to 0.1 ng/mL; the 50 percent relative binding sensitivity was seen at 32pg/20 microL/tube. Plasma volumes for the RIA, viz. 10 and 20 microL, did not influence the shape of standard curve, even though a slight drop in the counts was seen with higher plasma volumes. For the biological validation of the assay, three cyclic, three in early pregnancy, and two in late pregnancy mithuns were used. Blood samples collected at weekly intervals for 42 days, from all animals, were assayed for plasma progesterone. The peak level of progesterone was registered at day 14 (day 21 of sampling) of the estrous cycle and the lowest at estrus; the progesterone concentrations increased and decreased gradually as sampling time advanced, in early and late pregnancy, respectively, which confirm the biological validation of the RIA. The RIA avoids the troublesome and laborious plasma extraction procedures. In conclusion, the RIA developed for progesterone determination in mithun blood plasma is sufficiently reliable, simple, and sensitive enough to estimate progesterone in all physiological variations in mithun.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C(18) column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).     

Vasopressin in cerebrospinal fluid and plasma of man, dog, and rat

Arginine-8-vasopressin (AVP) levels were measured by a sensitive and specific radioimmunoassay (RIA) in plasma and cerebrospinal fluid (CSF) of three species man, dog, and rat (Wistar and the Brattleboro strain). Basal plasma values were 1.7 pg/ml in Wistar rat, and 2.4 pg/ml in dog. Pentobarbitone, used as anesthetic during collection of CSF from dog and rat, caused a significant rise of plasma AVP values in Wistar rats, but not in dogs. After withdrawal of CSF, the plasma AVP levels of Wistar rats were increased to 29.5 +/- 9.5 pg/ml, whereas the CSF levels from the same animals were 11.5 +/- 3.9 pg/ml. The response to the various stimuli was similar in Brattleboro rats, heterozygous for hereditary hypothalamic diabetes insipidus, and in Wistar rats. In Brattleboro rats, homozygous for hereditary hypothalamic diabetes insipidus, AVP was neither detectable in plasma nor in CSF. In dog and man, AVP levels in CSF samples were higher than in simultaneously obtained plasma samples. The possibility that AVP present in CSF, might be released directly from the synthetizing hypothalamic nuclei into the ventricular system is discussed.     

冷水游泳对去肾上腺大鼠下丘脑,垂体和血浆AVP含量的影响

ＡＶＰ主要合成于下丘脑的室旁核和视上核，正常情况下，每毫升血浆ＡＶＰ含量在１０ｐｇ以下，近年来的研究表明，疼痛、手术、应激等强刺激可引起ＡＶＰ大量释放，血浆中的ＡＶＰ含量可达１００ｐｇ以上。由于ＡＶＰ可使血管平滑肌收缩，并具ＣＲＦ样作用，可促进垂体释放ＡＣＴＨ，因此被认为是一种应激激素，而下丘脑－垂体－肾上腺皮质轴被认为是经典的应激系统。本工作旨在通过去除应激轴上的肾上腺这一环节，来验证ＡＶＰ是否     

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatographic assay for the novel bio-reductive anti-tumor agent RH1 in human and mouse plasma.

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the experimental anticancer agent, RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) which is currently being evaluated by the CRC phase I/II committee. A 500 mg amino propyl solid-phase extraction cartridge was used to isolate RH1 from human plasma. Analysis was performed on a reversed-phase chromatography system using a 15 cm cyanopropyl column and isocratic elution with a 10% methanol-90% water (double distilled) solution. The lower limit of quantitation for RH1 was found to be 0.00375 microg/ml (3.75 ng/ml+/-8.3%) in water and following extraction from plasma. Recovery of >80%(+/-11.9%) was achieved over a five-day validation study. This method was used to carry out pre-clinical studies in BDF mice (standard strain of hybrid mice) at three dose levels (2, 5 and 10 mg/kg of RH1 in 0.9% (w/v) saline via an intraperotoneal injection). Standard Version of PC Winnonlin pharmacokinetic modelling software was used to model the data. A none-compartmental model was used to describe the disposition of RH1 in mice plasma. RH1 was rapidly eliminated from plasma with a mean plasma clearance of 23.4 ml/min, mean volume of distribution of 321.6 ml and mean t(1/2) alpha and beta decays of 4.8 and 9.6 min, respectively. RH1 in human and mouse whole blood and plasma was found to be stable up to 2 h.     

A perifusion method for examining arginine vasopressin (AVP) release from hypothalamo-neurohypophyseal system

A perifusion method has been developed using rat hypothalamo-neurohypophyseal system (HNS) or neural lobe to investigate the control mechanism of arginine vasopressin (AVP) release. A specific radioimmunoassay (RIA) for AVP was developed to measure AVP in perifusion medium employing anti-AVP serum which was obtained by immunizing rabbits. At a final dilution of 1/12,000, the antiserum showed less than 0.66 and 0.01% cross reactivity with lysine-vasopressin and oxytocin, respectively. But it did not cross reacted with other peptide hormones. The lowest detectable level of vasopressin was 0.5 pg/tube. The intra-assay coefficient of variation averaged 10.4%. The dilution curve of perifused medium was well paralled to the standard curve of AVP assay. AVP release from HNS or neural lobe gradually declined to the stable level in 90-120 min after the initiation of perifusion. Good repeatability of the AVP release from neural lobe was recognized by repeated stimulation with 10 min perifusion of 60 mM KCl at every 60 min. HNS released AVP in dose related manner to the osmotic challenge of sodium or glucose, and AVP release was stimulated from HNS by prostaglandin E2, but not by dopamine. These results show that the perifusion methods using AVP-RIA is a useful method to examine the AVP release from HNS or neural lobe.     

Liquid chromatography-electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring.

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 microl) with diethyl ether using 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C8 column (Zorbax-Eclipse, 50x4.6 mm I.D.) using a steep methanol-water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r2=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at -20 degrees C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.     

Enzyme immunoassay for arginine vasopressin (AVP). 1. Enzyme labelling of AVP and its application for AVP standard curve

Enzyme immunoassay for arginine vasopressin (AVP) was developed in this study. Enzyme-labelling of AVP and then application of the enzyme-labelled AVP to make an AVP standard curve were done. Also, the anti-AVP antibody was obtained from the immunized rabbits. The enzyme (beta-D-galactosidase) was combined with AVP by using the N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide. The AVP-beta-D-galactosidase complex was examined for the competitive binding with AVP to the anti-AVP antibody. Then, the solid phase (polystylene bead) coupled with IgG fraction of anti-rabbit IgG serum (goat) was used to separate the antigen-antibody complex. The enzyme activity of this complex was measured to obtain an AVP standard curve. As a result, enzyme immunoassay for AVP described here was sufficiently sensitive and specific. Thus, this enzyme immunoassay could be applied for the determination of physiological levels of AVP in plasma. It is advantageous that the AVP-beta-D-galactosidase complex was stable for several years with respect to enzyme activity and immunological activity. The specific anti-AVP antibody with high titer was gained from the immunized rabbits using bovine thyroid globulin as a carrier protein.     

Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na125I by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interleukin-1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte-macrophage-colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml). The mean concentration of TNF in 24-h culture supernatants of human MNC exposed to different concentrations of lipopolysaccharide (LPS) was found to increase in dose-dependent fashion and then level off between 50 and 100 ng/ml. The concentrations of IL-1 beta and alpha detected by specific RIAs in these supernatants were between 0.2 and 19 ng/ml and 0.04 and 1 ng/ml, respectively. The amount of TNF produced by human MNC in vitro was determined in a cohort of 50 normal volunteers. Without exogenous stimuli, TNF concentrations were almost always below the detection limit; with 0.5 ng/ml LPS, the median concentration of TNF was 2 ng/ml, and with PHA the median was 3.8 ng/ml. In cultures performed in the presence of indomethacin significantly (p less than 0.005) more TNF was produced. Using this RIA, we could detect TNF in the circulation of mice injected with Hu rTNF. When plasma samples of patients with febrile illnesses were added directly to the RIA, TNF was not detectable, with the exception of patients with malaria. These studies demonstrate the range and sensitivity of LPS-induced and mitogen-induced production of immunoreactive TNF by human MNC in vitro without interference of similar cytokines in bioassays.     

Carbonylmetalloimmunoassay (CMIA) a new type of non-radioisotopic immunoassay. Principles and application to phenobarbital assay.

A new non-radioisotopic immunoassay procedure, which we have termed carbonylmetalloimmunoassay (CMIA), is described. The tracers used in this approach are organometallic carbonyl complexes that can be detected at femtomole levels (300-700 fmol) by Fourier transform infrared (FT-IR) spectroscopy. The validity of the technique has been tested in a phenobarbital assay using as the marker a cyclopentadienylmanganese (I) tricarbonyl (cymantrene) moiety, ethyl acetate extraction to separate the free and bound organometallic fractions, and FT-IR spectroscopy to detect the CO stretching modes of the organometallic label. Typical dilution and standard curves obtained with this CMIA procedure are presented. The method was of comparable sensitivity to a [14C] radioimmunoassay (RIA) for the detection of phenobarbital. A comparison of the results for phenobarbital assays by both CMIA and RIA showed that higher titres were obtained using the CMIA method. The standard curves suggest that CMIA is a reliable and reproducible immunoassay procedure for phenobarbital.     

急性脑出血患者血浆和脑脊液中精氨酸加压素含量测定及意义

本文应用放射免疫法,对30例急性脑出血患者和20例正常人的血浆和脑脊液中精氨酸加压素(AVP)含量进行测定。结果表明,患者血浆和脑脊液中AVP含量均明显高于对照组(p<0.01和p<0.001)。而出血量越多则血浆和脑脊液中AVP升高越明显。这提示,脑脊液中AVP含量的高低在一定程度上反映脑实质的损害程度和脑水肿的严重程度。对预后评估有着重要意义。     

Urinary excretion of aquaporin-2 in water metabolism disorders]

Water metabolism plays an essential role in the homeostasis of body fluids in animals and humans. It is regulated by arginine vasopressin (AVP), renal function and water drinking. Disorders of water metabolism result in an increase or decrease in a body water or fluid, which manifest as hyponatremia, hypernatremia, polyuria, dehydration or edema. In the pathogenesis of such pathological conditions AVP is either directly or indirectly involved. Aquaporin-2 (AQP-2) is an AVP-dependent water channel in renal collecting duct cells. Approximately 3% of AQP-2 is excreted into the urine, which is measurable by RIA or Western blot using a specific antibody against AQP-2. There was positive relationship between urinary excretion of AQP-2 (UAQP-2) and plasma AVP levels in normal subjects. UAQP-2 varied in a wide range under ad libitum water drinking. The level of UAQP-2 was one eighth less in patients with central diabetes insipidus than in normal subjects, and it was 2.8-fold greater in patients with water retention. A hypertonic saline infusion test manifested the difference in the UAQP-2 response to an increase in plasma osmolality between the patients with central diabetes insipidus and the normal subjects. Acute oral water load clarified the impaired water excretion and the persistent elevation of UAQP-2 in patients with water retention. Such increased UAQP-2 was linked to nonsuppressible levels of plasma AVP despite hypoosmolality. These results indicate that UAQP-2 is a useful marker to diagnose disorders of water metabolism.     

Radioimmunoassay for Retrovir, an anti-human immunodeficiency virus drug

A direct radioimmunoassay (RIA) for the quantitation of Retrovir (zidovudine, azidothymidine, AZT) in biological fluids has been developed. The assay is sensitive with an I50 value of about 30 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation that ranged from 1.77 to 8.65% for the standard curve with human plasma. Inter-assay precision and accuracy were within acceptable limits. The RIA was validated by comparing results obtained form the analysis of rat plasma samples by both this RIA and a high-performance liquid chromatography method. None of the crossreactivities recorded should interfere with the assay system. The affinity constant of the antibody chosen for use was 1.4 z 10(9) L/mol.