Evidence that atrial natriuretic peptide tissue extraction is not changed by large increases in its plasma levels induced by pacing in humans.

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.

Atrial natriuretic peptide is not degraded by the lungs in humans.

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.

Does a digoxin-like substance participate in vascular and pressure control during dietary sodium changes in patients with primary aldosteronism?

To evaluate the importance of an endogenous sodium pump inhibitor in the pathogenesis of low renin human hypertension, the urinary excretion of a digoxin-like immunoreactive substance (DLIS) was measured in eight patients with primary aldosteronism (n = 5, with adenomas) during two sequential 1-week periods of low- (20 mmol/l NaCl) and high- (200 mmol/l NaCl) sodium intake. DLIS excretion increased consistently during high-sodium intake while urinary aldosterone, plasma renin activity, cortisol and adrenocorticotropic hormone did not change. Although blood pressure showed a time-course parallel to that of the urinary DLIS, the blood pressure increments were not accompanied by evidence of vasoconstriction since forearm blood flow (plethysmographic technique) increased and forearm vascular resistances were reduced. Moreover, the reactivity of forearm arterioles to local norepinephrine was unchanged during the period of low- and high-salt intake, despite the fact that an endogenous sodium pump inhibitor should, supposedly, sensitize the responses to an adrenergic agonist. Finally, forearm vasoconstrictor responses to ouabain, a pharmacological Na+,K(+)-ATPase antagonist, were potentiated during the high-salt diet, a result not expected if an increased number of sodium pumps were occupied by an endogenous inhibitor. These results provide unequivocal evidence for a modulation by salt intake of the urinary excretion of a DLIS in patients with primary aldosteronism. This substance might participate in the regulation of body fluid volume in this syndrome and possibly in other physiological conditions. However, no evidence could be found for a cause--effect relationship between blood pressure and DLIS increments during high-salt intake, at least during the short-term course of the study.

Macro serum CK-BB in a woman with severe primary hypothyroidism.

We report the case of a woman with severe hypothyroidism and without concomitant myocardial damage, in whom elevated CK-MB values were measured by radioimmunological and enzymatic methods before and after thyroid replacement therapy. The patient's CK-MB activity was shown to be actually due to an atypical CK band between CK-MM and CK-MB (also termed "macro CK" or "idiopathic serum CK-BB").

Endogenous cardiac glycoside-like substances in newborns, adults, pregnant women and patients with hypertension or renal insufficiency.

In an attempt to confirm the presence of endogenous substances with cardiac glycoside-like activity, the biological and immunological cardiac glycoside-like activity was measured by a sensitive solid-phase radioimmunological assay (RIA), two radioreceptor assays (RRA), and a 86Rb uptake method in normal subjects and in some pathophysiological conditions characterized by sodium retention and volume expansion. Significant concentrations of digoxin-like immunoreactive substances (DLIS) were measured in plasma (or serum) of normal subjects while significantly higher levels were found in pregnant women, newborns and in patients with renal impairment, and in some with essential hypertension. Concentrations in urine of normal adults or newborns were several times higher than in plasma. The results obtained by RIA correlated with those obtained by RRA and 86Rb uptake methods. In 88 normal subjects, DLIS excretion rates (overnight urine collection) in men were significantly higher than in women (68.6 +/- 23.6 pg/min vs 50.9 +/- 21.0 pg/min, p less than 0.01). The DLIS excretion rates correlated with creatinine, Na and K urinary excretion rates, and also with the subjects' body weight, height, body mass index, and systolic blood pressure. These findings confirm the presence of endogenous substances with immunological and biological activity similar to cardiac glycosides in human body fluids and also confirm the hypothesis that these endogenous factors may be involved in fluid and electrolyte regulation in man. In addition, the present data indicate that urinary excretion of DLIS is dependent on body mass and renal glomerular filtration.

Elevated free cortisol plasma levels in patients with prostatic carcinoma undergoing treatment with estrogens.

The total cortisol concentration (TCC), the percentage of free cortisol (%FC) and the apparent free plasma cortisol concentration (AFCC) have been determined in plasma samples of 9 patients affected by prostatic carcinoma undergoing treatment with estrogens measured by an equilibrium dialysis system and a radioimmunological method. The values found in this group of patients were compared to those obtained in 9 untreated control patients and in normal healthy persons. The mean TCC values found in treated patients were significantly higher than in the controls, both in basal condition (circadian rhythm) and after dynamic tests, while the mean %FC values were significantly lower than the controls except for the results obtained after an ACTH stimulation test. The mean AFCC values in treated patients were elevated both in basal condition and after the dexamethasone suppression test (DST), but the difference between the two groups of patients was statistically significant only after DST. This study demonstrates that patients with prostatic carcinoma undergoing treatment with estrogens can have markedly increased free plasma cortisol levels with an impairment of the hypothalamus-pituitary-adrenal axis.

Modifications in the concentrations of circulating myoglobin after treatment with low doses of adriamycin.

The modifications in the concentration of circulating myoglobin have been studied by means of a radioimmunoassay in 15 cancer patients undergoing polychemotherapy including adriamycin. In 8 patients significant increases in myoglobin levels were found after injection of low doses of the drug (25-50 mg/m2). Moreover, a disturbance of the normal biorhythm of the protein was evident in 12 patients. Creatine kinase-MB was evaluated by means of a radioimmunoassay, but there was no relation between an increase in the isoenzyme and an increase in myoglobin. No ECG modifications were detected. These data indicate that the measurement of myoglobin may offer an indication of myocardial or skeletal muscle damage caused by adriamycin.

Critical evaluation of endothelins assay.

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)

Acute hypotensive effect of calcium antagonists and endogenous digitalis-like immunoreactivity in human essential hypertension.

Evidence has been provided on the increased presence, in essential hypertension, of endogenous digitalis-like factor(s) [DLIS, digoxin-like immunoreactive substance(s)] able to cross-react with antidigoxin antibodies and to inhibit the membrane-bound sodium-potassium pump. An inhibition of the sodium pump could lead, in smooth muscle cells, to an increase of intracellular calcium ions and to an increase of total peripheral resistances. In this study the relation between plasma levels of DLIS and the acute hypotensive effect of a calcium antagonist (nifedipine) has been evaluated in a group of borderline to severe hypertensive patients and in a control group of normotensive subjects. The results obtained confirm that the hypotensive effect of nifedipine is related to pretreatment blood pressure and show, only in hypertensive patients, a significant relation of DLIS with both pretreatment blood pressure and blood pressure decrement induced by nifedipine. These findings are compatible with a possible role of DLIS in modulating cellular calcium handling.

Evaluation and comparison of the analytical performances of two RIA kits for the assay of atrial natriuretic peptides (ANP).

We evaluated the analytical performance of two commercial RIA kits for the assay of plasma atrial natriuretic peptide (ANP), which use different antisera and tracers, in order to verify the most suitable RIA procedure for the assay of ANP in plasma samples. In particular, two different procedures for the purification of plasma were evaluated. The first one uses the extraction of plasma samples with Sep-Pack C18 cartridges and the second immunoextraction (C-terminal ANP-specific antibody bound to solid phase of Sepharose). The sensitivity and precision of the two RIA kits proved inadequate, being unable to provide an acceptably precise measurement of the plasma ANP concentrations in normal subjects. An improvement in precision and sensitivity was obtained by using "fresh" (or purified) preparations of the tracer and standard solutions, and by adding PEG to the B/F separation step. The direct assay (without preliminary purification) was not possible due to a serious overestimation of plasma levels due to the presence of interferences; on the other hand, the extraction step increased the imprecision and the complexity of the assay. Moreover, the extraction recovery of ANP added to plasma samples in the procedure using Sep-Pak cartridges is about 50-60%, while it was found to be almost complete (about 90%) in the immunoextraction procedure. Moreover, a comparison of the results obtained with the two RIA systems indicated that the antisera have comparable sensitivities, but quite different specificities; however, neither of the two RIA kits showed a completely satisfactory degree of sensitivity, precision and practicability.

Endogenous digitalis-like factors in human milk.

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.

Detection of hypothyroid myopathy by measurement of various serum muscle markers--myoglobin, creatine kinase, lactate dehydrogenase and their isoenzymes. Correlations with thyroid hormone levels (free and total) and clinical usefulness.

An accurate and close follow-up of serum levels of thyroid hormones and various muscle markers (myoglobin, creatine kinase and its isoenzymes CK-MB and CK-BB, lactic dehydrogenase, alpha-hydroxybutyric dehydrogenase, etc.) was carried out in 10 hypothyroid patients on replacement therapy. The main muscle markers, creatine kinase and myoglobin, were elevated respectively in 9 and 6 subjects. In 1 male significant CK-MB traces were measured, while in 1 woman significant CK-BB amounts were assayed. Significant correlations between patients' thyroid hormones and the levels of the muscle parameters were found. The rate of normalization of thyroid hormones and muscle markers in relation to replacement therapy was also studied. Myoglobin and creatine kinase have proved to be the best indicators of the hypothyroid myopathy, since they are sensitive for the early detection of muscle involvement due to the metabolic disorder and are closely correlated to the metabolic conditions of patients.

Free-cortisol assay by immunoextraction: comparison with an equilibrium dialysis procedure.

We describe a new method for directly determining the apparent free cortisol concentration in plasma samples by use of an antibody-coated test-tube RIA. Buffer-diluted plasma or standard serum is added to antibody-coated test tubes, incubated at 4 degrees C for 4 h, and the solutions are aspirated. 125I-labeled cortisol is added to each tube and incubated for 3 h at 4 degrees C. Then the insides of the tubes are washed and their radioactivities counted. The standard curve is in terms of free cortisol, the standard serum solutions having been measured with an equilibrium dialysis procedure. Plasma samples (n = 155) from normal subjects and from various patients, measured with the new immunoextraction method and the equilibrium dialysis technique, gave results that correlated well (r = 0.847, p less than 0.001). Results by this direct RIA also correlate well with the clinical adrenocortical status of patients for whom data on total plasma cortisol may be misleading. This simple, easy RIA is suited to be the routine method for free cortisol in plasma.

Circadian variations of serum myoglobin levels in normal subjects and patients with polymyositis.

Circadian variations in myoglobin levels were determined in 18 normal volunteers and in 10 patients with active polymyositis. In all the controls and in 9 of the 10 patients, serum myoglobin concentrations were highest at 9 am, fell significantly during the day to reach a nadir between 6 and 12 pm, remained constant until midnight, and then rose again at 3 am. The percent differences between the highest and lowest values ranged from 21-66%. This pattern was observed in both untreated patients and patients receiving 6-methyl-prednisolone, which was given either in a single dose at 8 am or in divided doses (8 am and 8 pm). These data demonstrate that the time of sample collection must be taken into account when myoglobin levels are used as a marker of muscle damage.

Progressively elevated levels of biologically active (free) cortisol during pregnancy by a direct radioimmunological assay of diffusible cortisol in an equilibrium dialysis system.

The measurement of free cortisol would be preferable with respect to the total hormone content, since it yields more reliable information about the plasma levels of the biologically active cortisol. We have developed a new method for the determination of the apparent free plasma cortisol concentration (AFCC) by means of direct radioimmunological measurement of dialyzed cortisol. The AFCC was measured in 23 plasma pools obtained from normal pregnant women at various gestational times and in 18 nonpregnant women. The mean AFCC was 19.5 +/- 7.1 ng/ml in pregnant women and 9.0 +/- 6.2 ng/ml in nonpregnant women (p less than 0.005). The percent of free cortisol (%FC) showed a progressive increase during pregnancy reaching the lower limits of the normal range at the third trimester. However, the mean of %FC was significantly lower (p less than 0.005) in pregnant women than in nonpregnant women. Our data show a progressive increase of biologically active cortisol during pregnancy.

Evaluation of a new radioimmunological assay for the determination of the B subunit of creatine kinase enzyme.

We used a new radioimmunological (RIA) kit for the assay of B subunit of creatine kinase enzyme (CK). This RIA system uses a specific antisera against the B subunit as ligant, human CK-BB labelled with 125I as tracer, and purified human CK-BB isoenzyme as standard. The mean (+/- SD) sensitivity obtained was 0.25 +/- 0.16 ng/tube and the between assay variability was about 9-10%. Serum levels of 113 normal subjects was not normally distributed. The 95% of values was found below 5 ng/ml. This new RIA is usefull in clinical practice when serum levels of CK-BB isoenzyme must be determined. This method is quickly and it is characterized by a good degree of precision, but the CK-MB isoenzyme cross-reacts for about 40% in this RIA system. Therefore, for the clinical diagnosis by means of this RIA it is necessary to rule out the concomitant elevations of serum CK-MB values.

Circadian rhythm of myoglobin serum concentrations in healthy subjects demonstrated by cosinor analysis.

In order to evidentiate whether a circadian rhythm of the myoglobin serum concentration is present in humans, we measured serially the myoglobin levels in blood samples of 21 healthy subjects (10 females and 11 males), at bed rest, by means of a sensitive and accurate radioimmunological method. The data obtained were then evaluated according to the single and mean cosinor analysis, to assess the presence of a significant circadian rhythm. Eighteen of the 21 subjects showed a significant circadian rhythm. The calculated equation for the mean cosinor was: Y = 19.12 + 4.09 cos (15t-128.24) and the F-value for the zero amplitude test was 19.9 (p less than 0.001). Our data demonstrate that circadian variations of myoglobin serum concentration are likely to occur in humans. These findings strongly emphasize the need to take the time of sample collection into account when employing serum myoglobin as a marker for muscle damage.