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1.
J Clin Endocrinol Metab ; 83(8): 2898-906, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9709966

ABSTRACT

In an attempt to identify and quantify the sites of atrial natriuretic peptide (ANP) degradation, particularly the lungs, a new tracer method to study ANP metabolism in vivo in humans was developed and applied to patients with left ventricular dysfunction. Thirteen male, normotensive, cardiac patients with different degrees of left ventricular myocardial involvement were enrolled in the study. The study protocol required constant infusion (3 patients) or bolus injection (10 patients) of 125I-labeled ANP just upstream of the right atrium and blood sampling from different sites (pulmonary artery, aorta, inferior vena cava, and femoral vein) during the hemodynamic study. Data analysis was based on a kinetic model consisting of three blocks in series (right heart, lungs and left heart, and periphery) supplied by the same plasma flow (plasma cardiac output). Plasma levels of native ANP were measured with a sensitive and specific immunoradiometric assay method. ANP values measured in the aorta (163.9 +/- 144.8 pg/mL, n = 80) were superimposable on those measured in the pulmonary artery (161.8 +/- 136.5 pg/mL, n = 80). Negligible extraction of 125I-labeled ANP was found in the lungs and left heart block (on average 0.08 +/- 3.92%), whereas the peripheral block extraction (46.2 +/- 7.8%) accounted for almost total hormone removal from the blood (whole body extraction was 46.4 +/- 6.6%). ANP metabolic clearance rate (3.11 +/- 1.48, range 1.4-6.8 L/min) declined with the progression of left ventricular dysfunction (plasma cardiac output 3.46 +/- 1.08, range 1.2-5.7 L/min), and a close correlation between metabolic clearance rate and cardiac output was evident. Our data suggest that lungs do not extract, or extract only very small amounts, of labeled ANP administered iv to patients with different degrees of left ventricular myocardial involvement, and whole body extraction of labeled ANP remains relatively stable with the progression of disease, and the large reductions in clearance values observed in our patients can be ascribed mainly to the reductions in cardiac output.


Subject(s)
Atrial Natriuretic Factor/metabolism , Lung/metabolism , Ventricular Dysfunction, Left/metabolism , Adult , Aorta , Atrial Natriuretic Factor/blood , Cardiac Output , Femoral Vein , Hemodynamics , Humans , Iodine Radioisotopes , Kinetics , Male , Middle Aged , Pulmonary Artery , Vena Cava, Inferior , Ventricular Dysfunction, Left/physiopathology
2.
J Endocrinol Invest ; 21(3): 170-9, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9591213

ABSTRACT

Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels increase in patients with heart failure with the progression of clinical symptoms and with the deterioration of hemodynamics; consequently, assay methods for these peptides may be useful in the follow-up of cardiac patients. Non-competitive immunoradiometric assay (IRMA) methods for ANP or BNP do not generally require preliminary extraction and/or purification of the plasma sample, and so may be more suitable than competitive immunoradiometric assay (RIA) methods for the routine assay of plasma peptide concentrations. We evaluated the analytical characteristics and clinical usefulness of two IRMAs for plasma ANP and BNP, to verify whether these methods may be considered suitable for the follow-up of patients with heart failure. Both methods are based on the solid-phase sandwich IRMA system, which uses two monoclonal antibodies prepared against two sterically remote epitopes of peptide molecule; the first antibody was coated on the beads solid-phase and the second was radiolabeled with 125I. Blood samples were collected from a brachial vein in ice-chilled disposable polypropylene tubes containing aprotinin and EDTA after the patient had rested for at least 20 min in the recumbent position. Plasma samples were immediately separated by centrifugation and stored at -20 C until assay. The IRMA methods showed a better sensitivity and a wider working range sensitivity (about 2 ng/l) than those of RIA methods. Moreover, the normal range found with these methods (ANP = 16.1 +/- 8.6 ng/l, 5.2 +/- 2.8 pmol/l, BNP = 8.6 +/- 8.2 ng/l, 2.5 +/- 2.4 pmol/l) was similar to that generally reported using the most accurate methods, such as the other IRMAs or RIAs, using a preliminary extraction and purification of plasma samples with chromatographic procedures. Our results obtained in patients with different degrees of heart failure indicate that plasma ANP and BNP increase with the progression of clinical symptoms (NYHA class) (ANOVA p < 0.0001). Indeed, circulating levels of ANP (R = -0.701, no. = 86) and BNP (R = -0.745, no. = 55) were significantly (p < 0.0001) and negatively correlated with the left ventricular ejection fraction values. Furthermore, a close curvilinear regression (R = 0.960, no. = 215) was found between ANP and BNP values, because plasma BNP progressively increases more than plasma ANP in patients with different stages of heart failure. In conclusion, IRMA methods are preferable for the measurement of plasma ANP and BNP for experimental studies and routine assay because they are more practicable, sensitive and accurate than RIA procedures. Finally, BNP assay appears to be better than ANP for discriminating between normal subjects and patients with different degrees of heart failure.


Subject(s)
Atrial Natriuretic Factor/blood , Heart Failure/blood , Myocardium/metabolism , Nerve Tissue Proteins/blood , Adult , Aged , Humans , Immunoradiometric Assay , Middle Aged , Natriuretic Peptide, Brain
3.
J Clin Endocrinol Metab ; 82(3): 884-8, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9062501

ABSTRACT

Atrial natiurectic peptide (ANP) is a cardiac hormone with a very short plasma half-life, which plays an important role in a variety of clinical conditions associated with an increase in pressure and/or volume overload on the heart. The MCR of the hormone is considered to represent a stable parameter, reflecting the uptake and degradation rate of ANP by the periphery, only scarcely affected by rapid oscillations of circulating levels. To evaluate the extent to which MCR is affected by rapid and large variations of circulating levels of the hormone, we measured MCR in five patients with different degrees of myocardial function (from normal to severely impaired), in whom changes in ANP levels were induced by atrial and/or ventricular pacing. Cardiac output was simultaneously measured by thermodilution to calculate whole body extraction of ANP. During constant i.v. infusion of [125I]ANP, the hormone MCR was determined both under basal conditions (at tracer equilibration, 20-30 min after the start of infusion) and during atrial and ventricular pacing. Pacing maneuvers, begun 50 min after the start of infusion, induced a marked and rapid increase in endogenous plasma ANP values in all patients (on the average, 3,7-fold compared to basal values; range, 1.8-5.68), whereas corresponding values of [125I]ANP only minimally changed. The MCR of ANP (3.62 +/- 1.06 L/min, mean +/- SD) slightly decreased (by repeated measures ANOVA, P = 0.0458) during atrial and ventricular pacing procedures (3.35 +/- 1.03 and 3.15 +/- 0.74 L/min, respectively), reaching a mean value of 88.7 +/- 9.0% compared to basal. The small decrease in MCR could be almost completely ascribed to hemodynamic factors; indeed, basal cardiac output (5.76 +/- 1.70 L/min) was found, on the average, to be slightly decreased during atrial and ventricular pacing (5.28 +/- 1.46 and 5.16 +/- 1.33 L/min, respectively), and so whole body extraction of the hormone, measured before pacing (50.0 +/- 12%), remains stable throughout the study period (50.4 +/- 10.6% and 49.6 +/- 10% during atrial and ventricular pacing, respectively). Our findings demonstrate that degradative mechanisms involved in ANP clearance are not saturable at least for acute elevations of ANP plasma levels up to 3-5 times the basal level.


Subject(s)
Atrial Natriuretic Factor/metabolism , Cardiac Pacing, Artificial , Adult , Aged , Atrial Function , Atrial Natriuretic Factor/blood , Cardiac Output , Female , Humans , Male , Metabolic Clearance Rate , Middle Aged , Ventricular Function
4.
Clin Chem ; 42(10): 1627-33, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8855146

ABSTRACT

We evaluated the analytical characteristics and clinical usefulness of a commercially available IRMA kit for measuring plasma concentrations of atrial natriuretic peptide (ANP) in healthy subjects and in patients with heart failure. The method uses two monoclonal antibodies prepared against sterically remote epitopes of the ANP molecule; the first antibody is coated on the solid-phase beads, and the second is radiolabeled with 125I. Fifty-nine healthy subjects and 77 patients with heart failure were studied. After subjects had rested 20 min in a recumbent position, blood samples were collected from a brachial vein into ice-chilled disposable polypropylene tubes containing aprotinin and EDTA. Plasma samples were immediately separated by centrifugation and stored at -20 degrees C until assay. The working range (CV <15%) was 10-2000 ng/L. The detection limit (2.13 +/- 0.91 ng/L) was similar to those reported for other IRMAs but was much better than those of RIAs. For healthy subjects, the results of this method (18.0 +/- 10.6 ng/L, range 4.7-63 ng/L, median 16.7 ng/L, n = 59) were similar to those generally reported for the most accurate methods, i.e., those using preliminary extraction and chromatographic purification of plasma samples. Measured plasma ANP was significantly associated with the severity of clinical symptoms, i.e., NYHA class (ANOVA, P <0.0001), and with the left ventricular ejection fraction (n = 62, r = 0.618, P <0.0001). Patients with severe heart failure showed greatly increased values (NYHA III-IV: 257.4 +/- 196.6 ng/L, n = 23).


Subject(s)
Atrial Natriuretic Factor/blood , Cardiomyopathy, Dilated/blood , Coronary Disease/blood , Immunoradiometric Assay/methods , Reagent Kits, Diagnostic , Adult , Aged , Aorta , Drug Stability , Female , Humans , Immunoradiometric Assay/statistics & numerical data , Male , Middle Aged , Posture , Pulmonary Artery , Reagent Kits, Diagnostic/statistics & numerical data , Reference Values , Sensitivity and Specificity , Vena Cava, Inferior
6.
Eur J Nucl Med ; 22(9): 997-1004, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7588954

ABSTRACT

In the present paper we evaluate the optimum chemical conditions for labelling atrial natriuretic peptide (ANP) and its metabolites and for preparing highly purified radiotracers which can be used for in vivo kinetic studies of ANP in humans. Synthetic alpha h1-28ANP and some hormone metabolites were iodinated with Na125I or Na131I by means of the lactoperoxidase (ANP) or the chloramine-T (ANP metabolites) technique. The biological activity of labelled ANP was tested by means of a binding study using mouse cardiac membranes. A high-performance liquid chromatography (HPLC) procedure was used to purify the labelled hormone and the principal labelled metabolites in venous plasma samples collected up to 50 min after the injection of 125I-labelled ANP from nine healthy men. The main ANP kinetic parameters were derived from the disappearance curves of the [125I]ANP, which were satisfactorily fitted by a biexponential function in all subjects. The main advantages of this tracer technique are: (1) high accuracy, allowing the identification of the metabolites produced in vivo under steady-state conditions after injection of the precursor (labelled hormone); (2) high sensitivity, allowing the detection of minimal quantities of metabolites (that cannot be identified on the basis of the integrated areas from the ultraviolet-absorbing peaks on HPLC); (3) high specificity, allowing the detection of possible in vitro artefactual generation of cleavage products of ANP using an internal labelled standard. Utilizing this tracer method, it was possible to estimate the principal parameters of ANP kinetics and also to plot the appearance curves of the labelled metabolites produced in vivo after the injection of the labelled precursor.


Subject(s)
Atrial Natriuretic Factor/metabolism , Iodine Radioisotopes , Adult , Amino Acid Sequence , Animals , Atrial Natriuretic Factor/chemistry , Chromatography, High Pressure Liquid , Humans , Isotope Labeling , Male , Mice , Molecular Sequence Data , Rats
7.
Aging (Milano) ; 7(2): 117-22, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7548261

ABSTRACT

The effects of two types of anti-aging dietary restrictions restrictions-60% diet restriction (DR) and every-other-day feeding (intermittent feeding, IF)-on age-related changes in atrial natriuretic factor (ANF) metabolism were studied in male Sprague Dawley rats by standard radioimmunological procedures and transmission electron microscopy. Both diet restrictions decreased plasma ANP (atrial natriuretic peptide) levels significantly (12-month-old rats: ad libitum fed controls 96 +/- 17.3 pg/mL, IF 63 +/- 4.8 pg/mL, DR 74 +/- 14.4 pg/mL; 24-month-old rats: AL 109 +/- 6.3 pg/mL, IF 75 +/- 8.9 pg/mL, DR 65 +/- 9.1 pg/mL). Dietary restriction prevented the age-related increase in ANP concentration in both the right atrium (12-month-old rats: AL 0.25 +/- 0.033 microgram/mg, IF 0.22 +/- 0.023 microgram/mg, DR 0.30 +/- 0.044 microgram/mg; 24-month-old rats: AL 0.39 +/- 0.037 microgram/mg, IF 0.10 +/- 0.015 microgram/mg, DR 0.07 +/- 0.011 microgram/mg) and left atrium (12-month-old rats: AL 0.23 +/- 0.033 microgram/mg, IF 0.13 +/- 0.019 microgram/mg, DR 0.17 +/- 0.035 microgram/mg; 24-month-old rats: AL 0.44 +/- 0.037 microgram/mg; IF 0.07 +/- 0.009 microgram/mg, DR 0.07 +/- 0.011 microgram/mg). Endocrine cardiocytes of diet-restricted senescent rats could be readily distinguished ultrastructurally from cells of ad libitum fed controls due to a higher number of beta-atrial specific granules of lower electrondensity. In conclusion, anti-aging diet restriction regimens fully prevented the age-related increase in the hormone store in atrial tissue, and lowered plasma ANP levels.


Subject(s)
Aging/metabolism , Atrial Natriuretic Factor/blood , Diet , Energy Intake/physiology , Age Distribution , Aging/blood , Animal Nutritional Physiological Phenomena , Animals , Body Weight , Cell Size/physiology , Heart Atria/cytology , Heart Atria/metabolism , Heart Atria/ultrastructure , Immunohistochemistry , Male , Microscopy, Electron , Organ Size , Radioimmunoassay , Rats , Rats, Sprague-Dawley
8.
Ann Ital Med Int ; 10(2): 107-12, 1995.
Article in Italian | MEDLINE | ID: mdl-7619649

ABSTRACT

In this study we compared the pressor, renal and endocrine effects of the physiological precursor of endothelial derived nitric oxide, L-arginine, with D-glucose, a substrate inactive on nitric oxide. Ten subjects with mild to moderate primary hypertension underwent infusion with either L-arginine (5 patients) or D-glucose (5 patients). The substances were infused over 25 min at equiosmolar rates, preceded and followed by a 25-min saline infusion. Blood pressure (BP) and heart rate were monitored at 3-min intervals; hormonal and humoral variables, inulin and para-aminohippurate clearance, and electrolyte excretion were measured at the end of each period at maximum diuresis. L-arginine and D-glucose brought about comparable increases in serum osmolality and similar hemodilution as compared with control saline. During L-arginine infusion, systolic and diastolic BP dropped by 16.6% and 11% respectively and recovered during the post-infusion period. Heart rate, plasma renin activity, and plasma norepinephrine did not change significantly. The percent BP decrease induced by L-arginine was significantly greater than that caused by D-glucose. Glomerular filtration rate remained stable, and renal plasma flow increased with both substances. However, only L-arginine stimulated markedly natriuresis, kaliuresis, and chloruresis. It also seemed to induce systemic acidosis, possibly as a consequence of hydrochloric acid generated during its metabolism. Circulating insulin, atrial natriuretic peptide, growth hormone, and glucagon levels increased, and plasma aldosterone remained unchanged during L-arginine infusion. During D-glucose infusion, insulin was stimulated and the other hormones were inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Arginine/pharmacology , Blood Pressure/drug effects , Endocrine Glands/drug effects , Hypertension/drug therapy , Hypertension/physiopathology , Kidney/drug effects , Aldosterone/blood , Arginine/administration & dosage , Atrial Natriuretic Factor/blood , Female , Glomerular Filtration Rate/drug effects , Glucagon/blood , Glucose/administration & dosage , Glucose/pharmacology , Growth Hormone/blood , Humans , Infusions, Intravenous , Insulin/blood , Male , Middle Aged , Monitoring, Physiologic
9.
J Endocrinol Invest ; 18(3): 194-204, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7615905

ABSTRACT

Using a tracer method, we evaluated, in vivo, the main turnover parameters and the main metabolic pathways of ANP in 10 normal subjects. HPLC was used to purify the labeled hormone and the principal labeled metabolites present in venous plasma samples collected at determined times after tracer injection. The main ANP kinetic parameters were derived from the disappearance curves of [125I] ANP, which were satisfactorily fitted by a biexponential function in all subjects. Newly produced ANP initially distributes in a large, plasma equivalent space (10.9 +/- 3.6 l/m2 body surface); the hormone rapidly leaves this space due to both degradation and to distribution in peripheral spaces. The mean residence time in the body (19.4 +/- 19.8 min) and the plasma equivalent total distribution volume (28.2 +/- 11.5 l/m2) indicate that ANP is also widely distributed outside the initial space in humans (circulating ANP is no more than 1/15 of the body pool). Metabolic clearance rate values were distributed across a wide range (from 740 ml/min/m2 to 2581 ml/min/m2, mean 1849 ml/min/m2), and were shown to strongly correlate (R = 0.962) with the daily urinary excretion of sodium. A complete separation of labeled ANP from its labeled metabolites was achieved by the HPLC technique; at least 3 different peaks due to labeled metabolites in vivo produced from the injected [125I]ANP1-28 were found. The first chromatographic peak eluted showed an identical elution time to monoiodotyrosine. At least two other peaks due to in vivo generated labeled metabolites were well identified in the chromatograms: one peak (coeluting with labeled COOH-terminal tripeptide, H-Phe-Arg-Tyr-OH) was eluted ahead and one (coeluting with labeled peptide fragments ANP7-28, ANP13-28, and ANP18-28) behind the elution peak of the labeled ANP. The peak of labeled tyrosine appearing in the plasma ranged between 3 and 5 min after tracer injection; the other two peaks of radioiodinated metabolites showed their highest activity in the first sample (1.5 min), suggesting an earlier occurrence of their peaks. These labeled metabolites seem to be intermediate peptides, between the intact circulating form of the hormone and the final labeled metabolite (tyrosine), which is the last amino acid of the peptide hormone, produced in vivo after injection of the tracer.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Atrial Natriuretic Factor/blood , Adult , Amino Acid Sequence , Humans , Iodine Radioisotopes , Kinetics , Male , Middle Aged , Molecular Sequence Data , Peptide Fragments/blood , Tyrosine/blood
10.
Eur J Clin Pharmacol ; 48(3-4): 195-201, 1995.
Article in English | MEDLINE | ID: mdl-7589041

ABSTRACT

The pressor, renal and endocrine effect of the physiological precursor of endothelial derived nitric oxide, L-arginine was compared, with a substrate inactive on nitric oxide, hypertonic D-glucose, in hypertensive patients. Ten mild-moderate essential hypertensives were assigned to either L-arginine (n = 5) or D-glucose (n = 5). Substances were infused over 25 min at equiosmolal rates preceded and followed by saline infusion for 25 min. Blood pressure and heart rate were monitored at 3-min intervals, while hormonal and humoral variables, inulin and paraaminohippurate clearance and electrolyte excretion were measured at the end of each period under conditions of maximal diuresis. L-arginine and D-glucose increased serum osmolality comparably and caused similar haemodilution to that with control saline. During L-arginine infusion, systolic and diastolic blood pressure decreased by 16.6% and 11%, respectively, and recovered in the postinfusion period. Heart rate, plasma renin activity, and plasma noradrenaline did not change significantly. The percent blood pressure decrement induced by L-arginine was significantly greater than that by D-glucose. Glomerular filtration rate was stable and renal plasma flow was increased by both substances. However, natriuresis, kaliuresis and chloruresis were markedly stimulated only by L-arginine, which also promoted the development of systemic acidosis, possibly as a consequence of hydrochloridric acid generated during its metabolism. Circulating insulin, atrial natriuretic peptide, growth hormone and glucagon levels were increased and plasma aldosterone was unchanged during infusion of L-arginine. Insulin was stimulated and the other hormones inhibited during infusion of D-glucose.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Arginine/therapeutic use , Blood Pressure/drug effects , Hypertension/drug therapy , Kidney/drug effects , Blood Proteins , Female , Humans , Insulin/metabolism , Male , Middle Aged , Osmolar Concentration , Time Factors , Urination/drug effects
12.
Aging (Milano) ; 6(3): 167-74, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7993924

ABSTRACT

The effects of age on atrial natriuretic factor (ANF) metabolism were studied in male Sprague Dawley rats by standard radioimmunological procedures, transmission electron microscopy, and ultrastructural morphometry. Plasma atrial natriuretic peptide (ANP) levels increased during growth (1-month-old rats: 64 +/- 8.7 pg/mL; 2-month-old rats: 105 +/- 8.1 pg/mL) and did not change thereafter. Immunoreactive (ir)-ANP concentration increased significantly in senescent rats, both in the right (12-month-old rats: 254 +/- 33 ng/mg; 24 month-old: 415 +/- 77 ng/mg) and left atrium (226 +/- 30 and 498 +/- 60 respectively). The ultrastructural morphological features of the endocrine cardiocytes of senescent rats were the presence of a few lysosomal structures and atrial specific granules of higher electron-density. Ultrastructural morphometry studies did not reveal any significant increase in the number or in the mean individual volume of atrial specific granules, compared with young adult rats. In conclusion, unlike younger rats, morphometric data in older and senescent rats show that atrial granularity may not necessarily change together with atrial ir-ANP contents; ir-hormone assay findings show that senescent rats have ANP plasma levels within the norm, and a much larger hormone store in atrial tissue.


Subject(s)
Aging/physiology , Atrial Natriuretic Factor/metabolism , Myocardium/cytology , Aging/metabolism , Animals , Atrial Natriuretic Factor/blood , Heart Atria/cytology , Heart Atria/growth & development , Heart Atria/metabolism , Male , Myocardium/metabolism , Myocardium/ultrastructure , Rats , Rats, Sprague-Dawley
13.
Int J Tissue React ; 16(2): 79-87, 1994.
Article in English | MEDLINE | ID: mdl-7960504

ABSTRACT

Endothelin(s) is (are) generally measured by highly-sensitive RIA (radioimmunoassay) or sandwich-EIA (enzyme immunoassay) methods after preliminary extraction and chromatographic purification using Sep-Pak C18 cartridges; however, there is no general consensus about the range of endothelin circulating levels in healthy subjects and patients. We have evaluated the analytical performance of two commercial RIAs for plasma endothelin-1 assay (supplied by Peninsula Laboratories, Belmont, California, and by Biomedica Gruppe, Biomedica Gesellschaft mbH, Vienna, Austria) in order to verify whether differences in extraction procedures, antibody affinities or standard preparations can explain the different results obtained by these two RIAs. The RIA kits tested in the present study showed some differences in the analytical performance; in particular, different antibody specificities have been observed. The concentration range of endothelin(s) assayed with an imprecision better than 15%, which can be considered the working range, was wider for the Peninsula than for the Biomedica kit (i.e. from 1.6 to 50 fmol/tube vs 0.7 to 10 fmol/tube), whereas the two RIA kits showed similar sensitivity (i.e. about 0.2 fmol/tube). Taking into account the working range and sensitivity of the two RIAs to measure with an acceptable error the samples of normal subjects and the most part of patients, > or = 3-ml volumes of plasma must be extracted by Sep-Pak C18 cartridges and then measured by RIA. Owing to the central role played by the endothelin superfamily in several pathophysiological conditions, it is important to have a reliable assay for the measurement of these peptides in biological fluids and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Endothelins/blood , Radioimmunoassay/methods , Radioimmunoassay/standards , Adult , Antibodies, Monoclonal/immunology , Antibody Specificity , Endothelins/immunology , Humans , Immunoenzyme Techniques , Infant, Newborn , Sensitivity and Specificity
15.
J Nucl Biol Med (1991) ; 37(2): 83-7, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8373838

ABSTRACT

An automated fluorometric enzyme immunoassay system for the determination of serum myoglobin has been recently developed. This method is based on the sandwich immunoassay and uses two mouse monoclonal antimyoglobin antibodies; the first one is complexed onto glass fiber paper and the second is conjugated to an enzyme alkaline phosphatase which reacts with the substrate 4-methylumbelliferyl phosphate to generate a fluorescent product. Using a dedicated automated apparatus the time to the first result is eight minutes, with additional values being produced at one-minute intervals (about 50 samples/hour). We compared the analytical performance of this fluorometric enzyme immunoassay with that of a RIA set up in our laboratory for the routine assay of serum myoglobin. The automated fluorometric enzyme immunoassay showed lower between-assay variability (CV = 4.7% vs 13.8%) and higher sensitivity (0.3 ng/mL vs 7.2 ng/mL) than the manual RIA. Moreover, the two immunoassays gave similar results when serum samples of normal subjects and patients with coronary artery disease with or without acute myocardial infarction (AMI) were assayed (fluorometric immunoassay = -0.7 + 0.851 RIA, r = 0.991, n = 137). In conclusion, the automated fluorometric enzyme immunoassay tested in the present study produces reliable clinical results with a rapid turnaround time and therefore can be recommended for use in the early detection of AMI in a laboratory of Coronary Care Unit.


Subject(s)
Immunoenzyme Techniques , Myoglobin/blood , Evaluation Studies as Topic , Humans , Radioimmunoassay
17.
J Nucl Biol Med (1991) ; 37(1): 33-7, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8329475

ABSTRACT

We evaluated the performance and analytical parameters of a one-step magnetic IRMA kit for the measurement of myosin in serum. The method uses two monoclonal antibodies selected for their high affinity to the heavy chains of human ventricular myosin. The first antibody is coupled to a magnetic solid phase and the second one is labeled with 125I. The working range of the IRMA (range of myosin concentrations measured with an imprecision < 10% CV) was 250-3600 microU/L and the sensitivity 20.8 +/- 7.2 microU/L. The between-assay variability, evaluated from replicate measurements in different runs of two serum pools was 14.6 CV% for the first pool (259.1 +/- 37.8 microU/L) and 14.3 CV% for the second pool (442.0 +/- 63.1 microU/L), respectively. To evaluate the clinical usefulness of myosin as a marker of myocardial cell damage, serum myosin was measured in patients with acute myocardial infarction (AMI) (n = 9) or subarachnoid hemorrhage (n = 20). The results obtained with the myosin assay were compared with those of two other markers considered specific for myocardial necrosis (CK-MB and myoglobin). In eight patients with AMI, serum myosin was elevated 24-36 hours after the onset of chest pain and reached a maximum at 4-7 days, returning to control levels at 8-11 days. The one remaining AMI patient showed two subsequent peaks in serum CK-MB and myoglobin concentrations (thus suggesting an extension of myocardial necrosis), the myosin concentrations reaching their peak only after 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Immunoradiometric Assay , Myocardial Infarction/pathology , Myosins/blood , Biomarkers , Female , Humans , Male
18.
J Endocrinol Invest ; 15(10): 727-34, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1491121

ABSTRACT

The effects of an altered thyroidal status on the levels of immunoreactive (ir-) atrial natriuretic peptide (ANP) in serum and in the right and left atria, as well as on the subcellular structures of atrial myoendocrine cells were explored in groups of male Sprague Dawley rats given the vehicle or triiodothyronine in the toxicological dose-range (50 micrograms/100 g bw/day) for 0,5, 1,2,4,7 or 14 days. Plasma levels of ir-ANP were 30% higher in T3-treated rats compared with controls at 0,5 and 1 day after hormone administration and then decreased to levels 30-40% lower than controls at days 2 and 4 to rise again above control values on day 7 and 14. Atrial ir-ANP levels decreased at first both in the right and in the left atria with different latencies (1 and 2 days, respectively) and rose back towards control levels by day 4. Changes in the numerical density of specific granules followed a parallel temporal pattern. An increased in the individual volume of the granules followed was also observed. Investigation into the circulatory effects of T3 administration showed that the heart rate was increased by hour 12 after hormone administration (simultaneously with the early rise in plasma ir-ANP levels) and that blood pressure was increased by day 2.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Atrial Natriuretic Factor/blood , Endocrine Glands/cytology , Myocardium/cytology , Thyroid Hormones/pharmacology , Animals , Atrial Natriuretic Factor/immunology , Blood Pressure/drug effects , Endocrine Glands/drug effects , Endocrine Glands/ultrastructure , Heart/drug effects , Heart Rate/drug effects , Immunohistochemistry , Male , Microscopy, Electron , Myocardium/ultrastructure , Organ Size/drug effects , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Thyroxine/blood , Triiodothyronine/blood
19.
Clin Chem ; 38(4): 504-6, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1314718

ABSTRACT

We measured the concentration of endogenous digitalis-like factors (EDLFs) in milk or colostrum of women during nursing on different days after delivery. EDLF concentrations were assayed by a solid-phase RIA involving antidigoxin antibodies and by a radioreceptor assay (RRA) involving human placenta Na+/K(+)-ATPase. The mean (SD) EDLF concentrations as measured by RIA were 35.6 (19.4) ng of digoxin equivalents per liter in milk samples (n = 37) and 61.3 (12.5) ng/L in colostrum samples (n = 5); the mean EDLF concentration as measured by RRA in milk samples (n = 11) was 573 (717) ng/L (range 0-2098). EDLF concentration in milk is greater than circulating concentrations in healthy adults but is comparable with serum concentration in the third trimester of pregnancy. In milk and serum samples (n = 8) collected at the same time, heating and (or) extracting with Sep-Pak C18 cartridges before the RIA produced significantly different EDLF values from those in untreated serum (P less than 0.001) and milk (P = 0.035). EDLF in milk appeared to be not bound or weakly bound to milk protein, as indicated by the fact that boiling did not increase the digoxin-like immunoreactivity.


Subject(s)
Blood Proteins/analysis , Digoxin , Milk, Human/chemistry , Saponins , Cardenolides , Colostrum/chemistry , Female , Humans , Lactation , Placenta/enzymology , Radioimmunoassay , Radioligand Assay , Reference Values , Sodium-Potassium-Exchanging ATPase/metabolism
20.
J Nucl Biol Med (1991) ; 36(1): 41-5, 1992.
Article in English | MEDLINE | ID: mdl-1333283

ABSTRACT

We measured by RIA the serum and urinary digoxin-like immunoreactivity (EDLF) in 8 subjects with severe obesity and in 10 healthy, non-obese individuals (as a control group), to evidentiate whether circulating and urinary levels of EDLF are increased in obesity. For each individual, we measured the mean EDLF on two sera collected consecutively on two successive mornings, between 8-9 a.m. and the daily urinary EDLF excretion. Every subject collected his/her 24 hour urine in 5 different timed fractions. For each urine fraction, we measured the excretion of EDLF, electrolytes (Na and K), and creatinine. In obese people, the mean serum digoxin-like immunoreactivity (no. 8, 27.3 +/- 8.7 ng/L de) was significantly higher (unpaired t test, p = 0.0002) than in the controls (no. 10, 12.0 +/- 7.3 ng/L de), whereas control and obese subjects had superimposable 24 hour EDLF urinary excretion. Urinary excretion of EDLF significantly changed throughout the day in normals, but not in obese people. In a multiple stepwise regression analysis, urinary K+ and Na+ significantly (p less than 0.01) contributed to the regression with urinary EDLF (EDLF = 76.9 + 0.67 K+ - 0.24 Na+; R = 0.601, no. 40) in obese individuals. The in vivo kinetic and metabolic pathways of EDLF are conceivably different in obese and normal subjects. A difference in the production rate, binding to plasma proteins and/or removal mechanisms could explain the findings of higher circulating levels with normal EDLF urinary excretion in obese persons.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Blood Proteins/metabolism , Digoxin , Obesity/blood , Saponins , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Adult , Cardenolides , Female , Humans , Male , Middle Aged , Obesity/urine , Radioimmunoassay
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