Effect of congestive heart failure on rate of atrial natriuretic factor release in response to stretch and isoprenaline.

STUDY OBJECTIVE: The aim was to investigate the pattern of release of atrial natriuretic factor induced by mechanical and adrenergic stimulation from atria of rats with or without congestive heart failure. DESIGN: Monocrotaline, a pyrrolizidine alkaloid, was given to rats to cause severe pulmonary hypertension, leading to a marked degree of right ventricular hypertrophy and failure. Measurements of noradrenaline and atrial natriuretic factor were performed in each cardiac chamber and in plasma. Right and left atria of control rats and rats with congestive heart failure were isolated and subjected to mechanical or adrenergic stimulation to study the in vitro release of atrial natriuretic factor. MATERIALS: Studies were performed on plasma, ventricles, and isolated right and left atria of 276 male Wistar rats, 80-100 g weight, with or without congestive heart failure. MEASUREMENTS AND MAIN RESULTS: In monocrotaline rats right and left ventricular concentrations of noradrenaline were significantly reduced. In the same rats concentrations of atrial natriuretic factor fell to 15.2% in the right atria and to 65.5% in the left atria. Whole heart content of atrial natriuretic factor was diminished, while plasma concentrations were increased sevenfold. Isolated hypertrophied right atria of failing hearts did not release atrial natriuretic factor in response to stretch or to isoprenaline (10(-9)M) and they were insensitive to the inotropic action of isoprenaline. On the other hand, non-hypertrophied left atria from the same animals released increased amounts of atrial natriuretic factor under basal conditions and after both stimuli, despite reduced tissue stores of the peptide. CONCLUSIONS: Heart failure may deplete cardiac stores of noradrenaline and atrial natriuretic factor, especially in hypertrophied chambers, and can result in a decrease in the release of atrial natriuretic factor from atrial tissue in response to mechanical and adrenergic stimulation.

New heteromyeloma cell lines for the production of human monoclonal antibodies.

The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.

Methodological effects on the quantitation of serum ferritin by radio- and enzymoimmunoassays.

Serum ferritin is normally quantitated by sensitive radio- or enzymoimmunoassays, which are based either on a competitive reaction between the unlabelled and labelled antigen (RIA), or on a non-competitive reaction between the sample and the labelled antibody (IRMA and ELISA). Both methods appear to be satisfactory for the clinical evaluation of serum ferritin. However, the effect of these methods on the quantitation of serum ferritin has never been carefully studied. This paper describes and compares five different immunoassays for the evaluation of serum ferritin: two competitive RIAs, one non-competitive IRMA and two non-competitive ELISAs. The same anti-ferritin antibody and ferritin standards were used for all the assays. The RIAs were found to be less sensitive than either the ELISAs or the IRMA, and all showed a similar degree of cross-reactivity with ferritin extracted from spleen and HeLa cells. A significant difference between the assays was found in the determination of serum ferritin: in fact the RIAs over-estimated serum ferritin by about 50% more than the IRMA, whereas the ELISAs under-estimated serum ferritin by about 15% less than the IRMA.

Immune response to opiates: new findings in heroin addicts investigated by means of an original enzyme immunoassay and morphine determination in hair.

In the early 1970's, some papers appeared reporting an immune response to opiates in animals treated with morphine and in heroin addicts, but further researches failed to confirm these results in humans. The aim of the present work is investigating with a newly developed enzyme immunoassay the existence of specific antibodies to morphine in a group of opiate chronic users, controlled by means of the toxicological analysis of hair. Twenty five opiate addicts inpatients for detoxication treatments were investigated for the presence of morphine specific antibodies and for the morphine content in hair, as a marker of addiction to opiates. Antibodies to morphine were investigated using an original ELISA method using a morphine-human serum albumin conjugate immobilized into the wells of polystyrene microtiter plates. Morphine determinations in hair were accomplished by a radioimmunologic screening followed by HPLC confirmation of positive results. The group of opiate users, in which all the subjects resulted positive for morphine content in hair, showed in the ELISA test an average D OD% value significantly higher than the control group (p < 0.001); in particular, 16 out of 25 addicts could be classified positive for anti-morphine antibodies, which were identified as IgM. Inhibition studies demonstrated Ka's for morphine ranging from 10(4) to 10(10) M-1 and a high cross reactivity for codeine. The presence of circulating antibodies specific to morphine in chronic users of opiates is strongly supported by the present findings.

Reversed-phase high-performance liquid chromatographic determination of cocaine in plasma and human hair with direct fluorimetric detection.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine with direct fluorimetric detection, particularly intended for the routine analysis of hair and blood samples, is described. Benzoylecgonine, eluting before cocaine in a completely resolved peak, is also detectable. Detection is based on the weak native fluorescence of cocaine and benzoylecgonine, depending on the benzene ring present in both molecules. Hair samples (20-200 mg) were incubated overnight in 2 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method; the dried residue reconstituted with 500 microliters of 0.05 M NaH2 PO4 (PH 5.2) was injected. Blood plasma samples (200 microliters) were mixed with 150 microliters of 0.1 M Na2 HPO4 (pH 8.9) and extracted with 5 ml of chloroform-2-propanol (9:1); the organic phase was evaporated and the residue dissolved and injected as above. Isocratic reversed-phase liquid chromatography was carried out on a column (150 x 4.6 mm I.D.) packed with spherical 5-microns poly(styrene-divinylbenzene) particles; the mobile phase was 0.1 M potassium phosphate (pH 3)-methanol-tetrahydrofuran (70:25:5). The excitation and emission wavelengths were set at 230 and 315 nm, respectively. Under the described conditions, cocaine eluted in a symmetrical peak with a capacity factor of about 5. The limit of detection was about 1 ng/ml (0.2 ng injected), with a signal-to-noise ratio of 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.

Effects of changes in intravascular volume on atrial size and plasma levels of immunoreactive atrial natriuretic peptide in uremic man.

To study the trigger for the release of atrial natriuretic peptide (ANP) in man, we measured the atrial areas (AA) by 2-D echocardiography, the total blood volume (TBV) by 131I-serum albumin and plasma immunoreactive ANP (i-ANP) concentrations by radioimmunoassay, after prior plasma extraction, for 10 dialyzed uremic patients. Measurements were made when the patients were volume-loaded or volume-depleted by isoosmotic ultrafiltration and again 48 h later, when they were again volume-loaded. Analysis of plasma extracts by high-performance gel permeation chromatography revealed that the greatest amount of the i-ANP fraction was a peptide eluting like human synthetic alpha-ANP. Ultrafiltration consistently decreased the TBV, while spontaneous regain of body-fluids caused TBV to rise to pre-ultrafiltration levels. Changes in TBV were closely related in time to changes in both right (RAA) and left (LAA) atrial area and in plasma i-ANP concentrations. Significant direct relationships were found between TBV and RAA, TBV and i-ANP and between both LAA and RAA and i-ANP. Furthermore, the decreases and the increases in TBV, RAA and LAA were closely correlated with changes in i-ANP. Multiple regression analysis, however, revealed that the changes in plasma i-ANP were mainly related to the changes in RAA, with little or no relationship to the changes in TBV or LAA. These findings are evidence for a positive feed-back between the level of intravascular filing volume, extent of atrial distention and amount of i-ANP released into the blood stream.

High-sensitivity low-cost methods for determination of cocaine in hair: high-performance liquid chromatography and capillary electrophoresis.

Free solution capillary electrophoresis and high performance liquid chromatography were applied to the analysis of cocaine in hair. Capillary electrophoresis conditions were as follows. Background electrolyte: 0.050 M borate, pH 9.2; capillary: bare silica, 40 cm long, 50 micrograms i.d.; potential: 15 kV; detection: UV at 238 nm wavelength. In addition, the separation was accomplished in a 50 cm x 75 microns capillary with an instrument with a photodiode array detector, recording on-line UV spectra of peaks from 200 to 400 nm. The isocratic high performance liquid chromatographic method with fluorimetric detection used 230 nm (ex.) and 315 nm (em.) wavelengths. Cocaine separation was carried out under conditions summarized as follows: column packed with spherical polystyrene-divinylbenzene 5 microns particles, mobile phase 0.1 M potassium phosphate (pH 3.0)/methanol/THF (70:25:5) at a flow rate of 0.5 ml/min and at room temperature. Liquid-liquid and solid-liquid sample preparation methods were used. Typically, levels of cocaine in hair as low as 0.15-0.30 ng/mg were detected by capillary electrophoresis, while HPLC allowed the determination of concentrations lower by one order of magnitude (0.015 ng/mg). Intra- and inter-assay precision data of the two techniques were comparable and characterized by relative standard deviations in the range from 3 to 7%. On the other hand, the sample throughput of capillary electrophoresis (7-10 injections/h) was higher than high performance liquid chromatography (2 injections/h). A good correlation of the results (r2 = 0.92) between the two techniques was observed in the assay of real cases.

[Standardized radioimmunologic determination of T 3]. / Dosaggio radioimmunologico standardizzato della T3

A method for detection of total T3 in sera is described. Reagents, incubation conditions and two methods of separation of free from bound hormone (charcoal and PEG) have been accurately studied. Particular attention has been paid to the reagent preparation and to the stability of lyophilized products over time. The results obtained show that: 1) ANSA works better than Merthiolate as splitter of TBG-T3 binding. 2) Measurements performed changing incubation temperature gave results not significantly different. 3) The separation method does not affect results. The method is extremely rapid, 1 h of incubation at 37degreesC or 4 h at 4degreesC. The calibration curve is expressed as a ratio of the activity bound to a different concentration of hormone and is described by a linear function with a correlation coefficient higher than 0.995. Clinical results are in accordance with those reported by the literature.

[Radioimmunological determination of circulating thyroid hormones]. / Dosaggi radioimmunologici degli ormoni tiroidei circolanti.

In our study of the thyroid function, the methodological aspects and clinical significance of TSH, T4, T3 and their respective free fractions (FT4 and FT3), are examined. The RIA of TSH is the best for the diagnosis of hypothyroidism and also for the evaluation of the effect of thyroid hormone substitution therapy. This, united with T4 and T3 assay, allows a total evaluation of the thyroid state. The new method, recently introduced, for direct assay of free fractions, using a chromatographic separation of Sephadex LH 20, is extremely useful for follow-up of patients with thyroid disfunction.

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor.

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm x 3 mm I.D.) in series, packed with 10 microns HEMA-S 1000 packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 microns), packed in a 30 mm x 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-microliters volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the "front" of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125-4 micrograms injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 micrograms injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).

Protein A, hydroxyapatite and diethylaminoethyl: evaluation of three procedures for the preparative purification of monoclonal antibodies by high-performance liquid chromatography.

Three rapid, reproducible and feasible monoclonal antibody purification procedures by means of high-performance liquid chromatography have been evaluated. The stationary phases employed were high-performance hydroxyapatite, high-performance Protein A and high-performance anion-exchange resin. The purity of the final immunoglobulin preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and, subsequently, by high-performance gel permeation chromatography. The immunoreactivity of purified monoclonal antibodies was determined by the radioimmunoassay method.

Methods for chromatographic determination of amanitins and related toxins in biological samples.

Methods for the chromatographic determination of amanitins, toxins of Amanita phalloides (Fr.), Link mushrooms and related toxins are reviewed; particular emphasis is given to high-performance liquid chromatographic methods. The main chemical and toxicological aspects are discussed, but the focus of the present review is on the analytical problems arising in a laboratory charged with the setting up of a procedure which can direct the appropriate clinical management of an intoxicated patient or solve a forensic case.

Capillary electrophoresis for the investigation of illicit drugs in hair: determination of cocaine and morphine.

Toxicological analysis of hair is becoming a popular method for investigating past, chronic use of illicit drugs. Several analytical methods using immunometry, chromatography and mass spectrometry have been reported. In this work, capillary electrophoresis was first used for the determination of illicit drugs, such as cocaine and morphine, in the hair of heroin and cocaine users. After rapid washing, hair samples were incubated overnight in 0.25 M HCl at 45 degrees C and the mixtures were extracted with ready-to-use Toxi-tubes A. The organic phase was evaporated and the residue dissolved in a suitable amount of electrophoresis buffer. Free zone capillary electrophoretic determinations of morphine, the main heroin metabolite, and cocaine were accomplished in 0.05 M borate buffer (pH 9.2) at a potential of 15,000 V, with UV detection at 214 and 238 nm, respectively. The use of the less selective wavelength of 200 nm allowed the simultaneous detection of both compounds. Efficient separations (up to 350,000 theoretical plates) and accurate and precise determinations (intra-day R.S.D.s in the range 3-5%) of cocaine and morphine in hair extracts were easily achieved. The analytical sensitivity was sufficient to determinate as little as 0.15 ng/mg of cocaine and morphine in hair using 100-mg samples. Interferences from more than 90 therapeutic drugs and drugs of abuse were excluded.

Kinetic analysis of TNF-alpha oligomer-monomer transition by surface plasmon resonance and immunochemical methods.

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 10(-5)/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-alpha, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Considerations on the sodium retention in nephrotic syndrome.

Renin-angiotensin-aldosterone system, plasma atrial natriuretic peptide (PANP), and blood volume (BV) have been investigated in 20 nephrotic patients with normal renal function and with (group 1; n = 12) or without (group 2; n = 8) sodium retention. Patients of group 1 had a plasma albumin (PALB) concentration < 1.7 g/dl, low BV and PANP levels, a reduced fractional excretion of lithium (FELi), and high plasma angiotensin II levels. Patients of group 2 had PALB > 1.7 g/dl, and the other parameters were normal. The spontaneous intake of dietary sodium was lower in group 1 than in group 2. In all patients the BV was directly correlated with PALB, and the plasma renin activity (PRA) was inversely correlated with both BV and PALB. A nonlinear inverse relationship was present between plasma aldosterone (PALD) levels and fractional excretion of sodium (FENa). The acute expansion of the BV in patients of group 1 normalized PRA, PALD, PAII, FENa, and FELi and increased PANP. The administration of spironolactone to the patients of both groups had variable effects on FENa, did not modify PRA and PALD, and reduced body weight, PANP, and FELi, thus suggesting that the reduction of BV induced by the drug increased the proximal reabsorption of sodium. Three additional patients who had sodium retention, PALB of 2.3-2.4 g/dl, normal PRA and PALD, elevated urinary excretion of aldosterone, and a slightly low PANP showed a spontaneous normalization of urinary aldosterone and PANP associated with natriuresis and weight loss, but thereafter urinary aldosterone increased, PANP decreased, and the sodium retention began again. Our data suggest that in nephrotic patients with severe hypoalbuminemia, contraction of BV plays a major role in promoting the sodium retention through the activation of compensatory hormonal mechanisms. On the other hand, when PALB is not severely reduced, the patients have normal BV, but they are very sensitive to small changes of BV which are better evidenced by modifications of the urinary excretion of aldosterone and PANP rather than by the profiles of PRA and PALD.

Immunoreactive "calcitonin-like" material in heroin addicts: varying reactivity with different antibodies.

High levels of immunoreactive calcitonin (iCT) in the blood of heroin addicts were previously reported. As it is well known that multiple forms of calcitonin exist in the blood and in tissues, the purpose of the present study was to investigate the immunological nature of the CT-like immunoreactive material found in the blood of these subjects. We investigated 25 addicts, who had been using heroin for more than one year and were hospitalized for a 2 week detoxication program. Blood samples were drawn at the start of the program (when the subjects were still on heroin) and after 5 and 12 days of abstinence from heroin. Twenty-five healthy subjects served as controls. We used 2 commercial RIA kits, calibrated against the same reference material (WHO 70-234), but employing different antisera. One antiserum substantially confirmed the previous findings of increased levels of calcitonin during heroin use, but the other seemed to exclude any change in the hormone concentrations. This suggests that the "calcitonin like" material found in heroin addicts contains some epitopes similar to those found in the calcitonin standard which are detected by the first antiserum. However it lacks other epitopes which are also present in calcitonin standard and which are recognized by the second antiserum. Therefore, this substance seems to be different from the standard human calcitonin 1-32. A possible involvement of a calcitonin analogue (precursor or metabolite) in the biochemical changes occurring during chronic opiate use is suggested.

A new method for free testosterone assay: clinical evaluation.

The Authors propose a new direct method for the determination of free Testosterone (F.Te). Our study was made following this method: 1) on a control group composed of 12 healthy men and 21 healthy women, and 2) on a group of 29 patients suffering from Polycystic Ovary Syndrome (PCOS) with clinical signs of hyperandrogenism. This pathological group presented acne and hirsutism in 95% of the cases. The Authors demonstrate how the determination of F.Te permits a 93.1% correct endocrinological diagnosis of hyperandrogenism.

Effect of reduction of blood volume on plasma immunoreactive atrial natriuretic factor concentrations in normal man.

Plasma immunoreactive atrial natriuretic factor (i-ANF) concentrations were measured in ten voluntary blood donors before and after acute blood loss induced after they had been supine for 20 min. Lying down caused i-ANF to increase significantly in all subjects. Bleeding (400 ml in 5 min) caused i-ANF to decrease to values significantly lower than either those observed before haemorrhage or those recorded in the upright position. These results provide evidence that in normal man acute changes in the intravascular volume, although limited within physiological limits, are able to induce opposite changes in i-ANF release.