Plasma concentrations of atrial natriuretic peptide in various diseases

Using a radioimmunoassay for atrial natriuretic peptide (ANP) we studied plasma concentrations of immunoreactive ANP in order to investigate the pathophysiological role of ANP in patients with various diseases. Plasma ANP levels were elevated in patients with congestive heart failure (394 +/- 260 pg/ml, n = 8) and chronic renal failure (219 +/- 86 pg/ml, n = 11). In patients undergoing hemodialysis plasma ANP levels were markedly high and decreased after hemodialysis from 433 +/- 166 pg/ml to 204 +/- 92 pg/ml (n = 11). ANP was removed from blood to dialysate (21 +/- 13 pg/ml of dialysate, n = 6, dialysate flow: 500 ml/min). Plasma ANP level was conversely correlated with creatinine clearance (r = -0.812, p less than 0.001) in patients with renal diseases (n = 29). In patients with atrial fibrillation, pace maker implantation, lung disease, chronic glomerulonephritis, nephrotic syndrome, essential hypertension, liver disease and cerebrovascular disease, plasma ANP levels were not significantly different from those in normal subjects (70 +/- 32 pg/ml, n = 28). These results suggest that ANP may be a circulating hormone playing pathophysiological roles in congestive heart failure and chronic renal failure.     

Maternal and fetal atrial natriuretic peptide levels, maternal plasma renin activity, angiotensin II, prostacyclin and thromboxane A2 levels in normal and preeclamptic pregnancies.

To clarify the possible role of elevated atrial natriuretic peptide (ANP) in the pathophysiology of preeclampsia, we measured ANP, renin activity (PRA), angiotensin II (Ang II), TXB2 (a stable metabolite of TXA2) and 6-keto-PGF1 alpha (a stable end product of PGI2) concentrations in the plasma of 19 normal pregnant women and 35 severe preeclamptic patients at term. Plasma ANP levels in the preeclamptic patients (n = 35, 71.5 +/- 3.8 pg/ml, mean +/- S.E.) and also umbilical plasma ANP (n = 35, 83.0 +/- 4.2 pg/ml) were significantly (p less than 0.01) higher than those of normal pregnant women plasma (n = 19, 58.7 +/- 3.7 pg/ml) and umbilical plasma (n = 19, 47.6 +/- 4.7 pg/ml). There was a significant (p less than 0.01) positive correlation between maternal ANP levels and fetal ANP levels (n = 54, r = 0.44). Plasma PRA and 6-keto-PGF1 alpha levels in preeclampsia were significantly (p less than 0.05) lower than those of normal pregnancy. The ratio of 6-keto-PGF1 alpha/TXB2 in preeclampsia was significantly (p less than 0.01) lower than that of normal pregnancy as we reported previously. There was no significant correlation between plasma ANP level and plasma PRA, Ang II, plasma TXB2 and 6-keto-PGF1 alpha concentrations. Moreover there was no significant correlation between plasma ANP level and the severity of preeclampsia. These data suggest the possibility of a transplacental crossing of ANP secreted by feto-placental unit, which might be, at least in part, responsible for the high ANP levels observed in preeclampsia. The ANP in preeclampsia is not related directly to hypertension, but it may play a substantial role in the regulation or normalization of blood volume and vascular reactivity.     

The circulating levels of cardiac natriuretic hormones in healthy adults: effects of age and sex.

In order to study the relationships between sex hormones, aging, and circulating levels of cardiac natriuretic peptides and to define reference values for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) assays, we measured the plasma levels of cardiac natriuretic peptides in a large group of healthy adults divided according to age and sex. We studied 216 healthy subjects of both sexes (109 men and 107 women) with age ranging from 20 to 77 years (mean 43.2+/-14.8 years). All subjects were non-obese and had normal arterial blood pressure; they were free from acute diseases, including asymptomatic heart disease. Highly sensitive and specific IRMA methods were used to measure plasma ANP and BNP. The mean ANP value in healthy adult subjects of both sexes was 17.8+/-10.9 pg/ml with no significant difference between men (16.7+/-10.0 pg/ml) and women (18.8+/-11.7 pg/ml). The mean BNP value in healthy adult subjects of both sexes was 9.9+/-9.0 pg/ml with a significant difference (p<0.0001) between men (7.7+/-7.1 pg/ml) and women (12.2+/-10.2 pg/ml). There was a weak linear relationship between age and either ANP (r=0.350, p<0.0001) or BNP (r=0.254, p=0.0002) values. When the circulating levels of cardiac natriuretic hormones, and age and sex were analyzed by multiple stepwise regression analysis, both age and sex significantly and independently contributed to the regression. Our study indicates independent positive effects of aging and female sex hormones on ANP and BNP levels in healthy adult subjects. These effects should be taken into account in the calculation of appropriate reference values for cardiac natriuretic hormones.     

Cardiac peptide stability, aprotinin and room temperature: importance for assessing cardiac function in clinical practice

Brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and N-terminal ANP are good research indices of the severity of heart failure. The stability of these peptides at room temperature has become an important factor in assessing their use as indicators of cardiac function in routine clinical practice. Inhibitors such as aprotinin are routinely added in the blood collection process, but may provide no benefit in sample collection and routine clinical practice. We assessed the stability of BNP, ANP and N-terminal ANP in blood samples collected in either the presence or the absence of the protease inhibitor aprotinin. Blood, either with or without aprotinin, was processed immediately (initial; 0 h) and after blood samples had been left for 3 h, 2 days or 3 days at room temperature. These times were chosen to reflect processing in a hospital outpatient clinic (2-3 h), or when posted from general practice (2-3 days). Initial plasma BNP, ANP and N-terminal ANP levels in the absence of aprotinin were 28.2+/-5.4, 44.2+/-7.9 and 1997+/-608 pg/ml respectively, and were not significantly different from initial values in the presence of aprotinin (29.0+/-5.9, 45.2+/-8.0 and 2009+/-579 pg/ml respectively). After 3 h at room temperature, there was a significant fall in ANP in the absence of aprotinin (36. 7+/-7.9 pg/ml; P<0.005), but not in the presence of aprotinin (41. 2+/-7.6 pg/ml). Both BNP and N-terminal ANP were unchanged in either the absence (BNP, 27.6+/-5.5 pg/ml; N-terminal ANP, 2099+/-613 pg/ml) or the presence (BNP, 29.4+/-5.6 pg/ml; N-terminal ANP, 1988+/-600 pg/ml) of aprotinin. After 2 days at room temperature, ANP had fallen significantly in both the absence (16.9+/-3.4 pg/ml) and the presence (24.0+/-5.0 pg/ml) of aprotinin compared with initial values, and there was a significant difference in ANP levels in the absence and presence of aprotinin (P<0.001). ANP levels had decreased further after 3 days at room temperature, to 11.9+/-3.4 pg/ml (no aprotinin) and 20.3+/-5.0 pg/ml (aprotinin added); these values were significantly different (P=0.002). In contrast, there was no change in the levels of BNP or N-terminal ANP after 2 or 3 days at room temperature, in either the absence or the presence of aprotinin. These studies indicate that aprotinin adds little benefit to the stability of cardiac peptides at room temperature. Blood samples for BNP and N-terminal ANP measurement used as a test of heart function in hospital clinics and by general practitioners in the community could be taken into blood tubes containing only EDTA as anticoagulant and without the additional step of adding the routinely used inhibitor aprotinin.     

Changes in the plasma levels of atrial natriuretic peptides during mineralocorticoid escape in man

Plasma levels of atrial natriuretic peptide (ANP) were measured by radioimmunoassay in eight normal healthy volunteers before and during mineralocorticoid escape. Mean plasma ANP on a fixed sodium intake before fludrocortisone was 6.5 +/- SEM 1.1 pg/ml. Within 24 h of fludrocortisone administration there was a significant increase in plasma ANP which continued to increase daily reaching a plateau by day 4 (14.9 +/- 2.4 pg/ml) to day 7 (15.1 +/- 2.6 pg/ml). The rise in plasma ANP was closely related to the amount of sodium retained during the fludrocortisone treatment and the sodium 'escape' occurred by days 4 to 7. These results support the concept that ANP could play an important hormonal role in over-coming the sodium-retaining effects of mineralocorticoids in man.     

Immunoreactive N-terminal pro-atrial natriuretic peptide in human plasma: plasma levels and comparisons with alpha-human atrial natriuretic peptide in normal subjects, patients with essential hypertension, cardiac transplant and chronic renal failure

1. Plasma levels of immunoreactive N-terminal pro-atrial natriuretic peptide (N-terminal ANP) have been measured in 25 normal subjects, 29 patients with essential hypertension, six cardiac transplant recipients, seven patients with dialysis-independent chronic renal failure and 11 patients with haemodialysis-dependent chronic renal failure. Plasma was extracted on Sep-Pak cartridges and N-terminal ANP immunoreactivity was measured using an antibody directed against pro-ANP (1-30). 2. Plasma levels of N-terminal ANP (means +/- SEM) were 235.3 +/- 19.2 pg/ml in normal subjects and were significantly raised in patients with essential hypertension (363.6 +/- 36.3 pg/ml), in cardiac transplant recipients (1240.0 +/- 196.2 pg/ml), in patients with chronic renal failure not requiring dialysis (1636.6 +/- 488.4 pg/ml) and patients with chronic renal failure on maintenance haemodialysis (10336.1 +/- 2043.7 pg/ml). 3. There were positive and significant correlations between the plasma levels of N-terminal ANP and alpha-human ANP (alpha-hANP) with individual correlation coefficients of 0.68 within the normal subjects, 0.47 in patients with essential hypertension, 0.78 in patients with dialysis-independent chronic renal failure and 0.68 in patients with haemodialysis-dependent chronic renal failure (P less than 0.05 in every case).(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic peptide in human plasma--comparison of radioreceptor versus radioimmunoassay

A sensitive and specific procedure for the measurement of atrial natriuretic peptide (ANP) in human plasma by radioreceptor assay, using bovine adrenal membranes treated with Triton-X-100, is described. Plasma levels (mean +/- SEM) of ANP in healthy subjects on a normal sodium intake were 8.4 +/- 1.4 pg/ml and could be modified by changes in sodium intake with increases in sodium intake being associated with higher levels. Mean plasma ANP was approximately 2-fold higher in patients with essential hypertension and 4-fold higher in patients with cardiac or renal disease. The values obtained were comparable in magnitude to those obtained by radioimmunoassay and there was a strong correlation (r = 0.94; p less than 0.001) between the values obtained by radioimmuno- and radioreceptor-assay. These results suggest that circulating ANP corresponds to the biologically active peptide and point to an important role of the atrial peptides in the control of sodium balance.     

Analytical performance and diagnostic accuracy of a fully-automated electrochemiluminescent assay for the N-terminal fragment of the pro-peptide of brain natriuretic peptide in patients with cardiomyopathy: comparison with immunoradiometric assay methods for brain natriuretic peptide and atrial natriuretic peptide.

We evaluated the analytical performance of a fully-automated electrochemiluminescence "sandwich" immunoassay method for the N-terminal fragment of the pro-peptide of brain natriuretic peptide (BNP). We then compared the diagnostic accuracy of this method in discriminating between normal subjects and patients with cardiomyopathy with that found with two previously described immunoradiometric assay methods for the assay of atrial natriuretic peptide (ANP) and BNP. We studied 193 consecutive patients (mean age 64.4 +/- 12.3 years, range 20-89 years, including 56 women and 137 men) with chronic cardiomyopathy and a group of 85 healthy subjects (mean age 52.3 +/- 12.0 years, 42 women and 43 men, range 20-79 years). N-terminal fragment of proBNP1-76 (NT-proBNP) was measured with a fully-automated "sandwich" electrochemiluminescence method using an Elecsys 2010 analyzer, while ANP and BNP were measured with immunoradiometric assay methods. The low detection limit of the NT-proBNP assay was 4.2 pg/ml (0.50 pmol/l), while the functional sensitivity was 22 pg/ml (2.60 pmol/l) with a working range (imprecision profile < or = 10% coefficient of variation) extended up to about 30 000 pg/ml (3540 pmol/l). Healthy women (64.3 +/- 41.6 pg/ml, 7.59 +/- 4.91 pmol/l) showed significantly higher values than men (46.9 +/- 30.9 pg/ml, 5.53 +/- 3.64 pmol/l, p = 0.0118). Moreover, age and sex were significantly and independently related to the NT-proBNP values in healthy subjects, as assessed by a multiple linear regression analysis (R = 0.389, F-value = 7.316, P-value = 0.0012). As expected, the NT-proBNP values of patients with cardiomyopathy were significantly higher than those of normal subjects and progressively increased with the severity of heart failure. The respective diagnostic accuracy of the ANP, BNP and NT-proBNP assays in discriminating between the group of normal subjects and that of patients with cardiomyopathy was tested by the response operating characteristic curve analysis. Our data indicated that the NT-proBNP assay is significantly better than either of the ANP or BNP immunoradiometric assays in discriminating affected patients from healthy subjects, especially when only patients with mild disease severity (New York Heart Association class I and II) are considered.     

Plasma levels of atrial natriuretic peptide in patients with borderline and essential hypertension

Plasma levels of atrial natriuretic peptide (ANP) were measured in outpatients with borderline hypertension (n = 15) and essential hypertension (n = 13) and in normotensive subject (n = 11). There were no significant differences among the three groups in age, serum protein, albumin, or electrolyte levels, plasma renin activity (PRA), or plasma concentrations of aldosterone and cortisol. The plasma ANP levels in the normotensive, borderline hypertensive, and essential hypertensive subjects were 36 +/- 6 pg/ml (mean +/- S.E.), 64 +/- 11 pg/ml, and 82 +/- 14 pg/ml, respectively. The levels in the essential hypertensive subjects were significantly (p less than 0.05) higher than those in the normotensives. In both borderline and essential hypertensives (n = 28), the plasma ANP levels were significantly correlated positively with systolic blood pressure (r = +0.385, p less than 0.05), and negatively with PRA (r = -0.484, p less than 0.05) and serum total calcium (r = -0.516, p less than 0.01). These results suggest that the elevation of circulating ANP in hypertensives is involved in the pathogenesis of hypertension.     

HEMODYNAMIC, RENAL, AND ENDOCRINE EFFECTS OF 4- H INFUSIONS OF HUMAN ATRIAL NATRIURETIC PEPTIDE IN NORMAL VOLUNTEERS

A synthetic human atrial natriuretic peptide of 26 aminoacids [human (3-28)ANP or hANP] was infused into normal male volunteers. Six subjects were infused for 4 h at 1-wk intervals with either hANP at the rate of 0.5 or 1.0 microgram/min or its vehicle in a single-blind randomized order. Human (3-28)ANP at the dose of 0.5 microgram/min raised immunoreactive plasma ANP levels from 104 +/- 17 to 221 +/- 24 pg/ml (mean +/- SEM), but it induced no significant change in blood pressure, heart rate, effective renal plasma flow, glomerular filtration rate, or renal electrolyte excretion. At the rate of 1.0 microgram/min, human (3-28)ANP increased immunoreactive plasma ANP levels from 89 +/- 12 to 454 +/- 30 pg/ml. It reduced effective renal plasma flow from 523 +/- 40 to 453 +/- 38 ml/min (P less than 0.05 vs. vehicle), but left glomerular filtration rate unchanged. Natriuresis rose from 207 +/- 52 to 501 +/- 69 mumol/min (P less than 0.05 vs. vehicle) and urinary magnesium excretion from 3.6 +/- 0.5 to 5.6 +/- 0.5 mumol/min (P less than 0.01 vs. vehicle). The excretion rate of the other electrolytes, blood pressure, and heart rate were not significantly modified. At both doses, human (3-28)ANP tended to suppress the activity of the renin-angiotensin-aldosterone system. In 3 additional volunteers, the skin blood flow response to human (3-28)ANP, infused for 4 h at the rate of 1.0 microgram/min, was studied by means of a laser-doppler flowmeter. The skin blood flow rose during the first 2 h of peptide administration, then fell progressively to values below baseline. After the infusion was discontinued, it remained depressed for more than 2 h. Thus, in normal volunteers, human (3-28)ANP at the dose of 1.0 microgram/min produced results similar to those obtained previously with rat (3-28)ANP. It enhanced natriuresis without changing the glomerular filtration rate while effective renal plasma flow fell. It also induced a transient vasodilation of the skin vascular bed.     

Increased secretion of atrial natriuretic polypeptide from the left ventricle in patients with dilated cardiomyopathy.

To examine whether atrial natriuretic polypeptide (ANP) is released from the left ventricle in patients with dilated cardiomyopathy (DCM) we measured plasma ANP level in the aortic root (Ao), the anterior interventricular vein (AIV), the great cardiac vein (GCV), and the coronary sinus (CS) in 11 patients with DCM and 18 control subjects. Plasma ANP levels in Ao, AIV, GCV, and CS were 454 +/- 360, 915 +/- 584, 1,308 +/- 926, and 1,884 +/- 1,194 pg/ml, respectively, in the patients with DCM and 108 +/- 42, 127 +/- 55, 461 +/- 224, and 682 +/- 341 pg/ml, respectively, in the control subjects. There was no significant difference in the plasma ANP levels between Ao and AIV in the control subjects. On the contrary, there was a significant (P less than 0.001) step-up in plasma ANP levels between Ao and AIV in patients with DCM. Thus, the difference in ANP levels between Ao and AIV was significantly increased in patients with DCM as compared with the control subjects (461 +/- 248 vs. 19 +/- 59 pg/ml, P less than 0.001). The difference in ANP levels between Ao and CS was also significantly increased in patients with DCM as compared with the control subjects (1,429 +/- 890 vs. 577 +/- 318 pg/ml, P less than 0.001). We conclude that ANP is released in increased amounts into the circulation from the left ventricle as well as from the heart as a whole in patients with DCM.     

Effects of angiotensin-converting enzyme inhibition in diabetic rats with reduced renal function.

To test the effect of converting enzyme inhibition (CEI) on diabetes, with or without renal insufficiency, we studied streptozotocin-induced diabetic rats, with or without reduced renal mass, which were treated with insulin in sufficient amounts to maintain glucose values in the mild to moderately hyperglycemic range. We found that diabetes increased glomerular filtration rate (GFR) (inulin clearance, 2.3 +/- 0.5 ml/min vs 1.9 +/- 0.1 ml/min; p < 0.05) and blood pressure (137 +/- 15 mm Hg vs 116 +/- 6 mm Hg; p < 0.05) but did not increase plasma atrial natriuretic peptide (ANP) values, when compared with control rats (72 +/- 38 vs 68 +/- 24 pg/ml). CEI decreased GFR and blood pressure to control values. In rats with diabetes and concomitantly reduced renal mass, hypertension, elevated ANP values, proteinuria, and glomerulosclerosis were prominent features. CEI was associated with reduced blood pressure (172 +/- 17 mm Hg vs 138 +/- 15 mm Hg; p < 0.05), without a concomitant decrease in GFR (1.1 +/- 0.1 ml/min vs 1.1 +/- 0.1 ml/min). Further, CEI reduced the elevated ANP values (140 +/- 34 pg/ml vs 66 +/- 19 pg/ml; p < 0.05) to those of control rats. CEI reduced proteinuria by 50% and ameliorated the histopathologic changes. In separate experiments, rats with 5/6th nephrectomy and hypertension but without diabetes were also found to have elevated ANP levels that decreased to control values with CEI. The data speak for a renal protective effect of angiotensin I-converting enzyme inhibition in this model but do not support a specific role for ANP in the model of diabetes with concomitantly reduced renal mass.     

Atrial natriuretic peptide in human essential hypertension

We measured the circulating levels of atrial natriuretic peptide (ANP) in 62 patients with untreated uncomplicated essential hypertension and in 30 normotensive subjects. In the hypertensive patients, mean systolic and diastolic blood pressures were 148 and 101 mm Hg, respectively, and the mean heart rate was 73 beats/min. ANP concentrations were not elevated in the hypertensive group but were actually decreased slightly over those of the control group (27.4 +/- 1.8 pg/ml versus 35.3 +/- 2.4 pg/ml [P less than 0.02]). No relationship was found between ANP levels and diastolic blood pressure, plasma renin activity, urinary sodium excretion, or serum creatinine level. In 8 of the 62 patients with essential hypertension, 6 weeks of treatment with a dihydropyridine calcium channel blocker, nitrendipine, significantly reduced plasma ANP levels from 28.6 +/- 4.3 pg/ml to 18.7 +/- 1.8 pg/ml (P less than 0.05). In 17 additional patients treated with the hypotensive agent ketanserin, ANP levels were not significantly reduced after treatment. Thus, this study demonstrates that circulating plasma ANP levels are not increased but are slightly decreased in patients with uncomplicated essential hypertension in comparison with normotensive subjects. Furthermore, antihypertensive treatment with a calcium channel antagonist reduced plasma levels of ANP.     

The relationship between atrial granularity and circulating atrial natriuretic peptide in hamsters with congestive heart failure

The BIO 14.6 strain of hamster is a model of familial cardiomyopathy complicated by congestive heart failure, sodium retention, and edema. In previous studies, bioassay techniques have demonstrated that the cardiac content of atrial natriuretic peptide (ANP) is reduced in these animals. On the basis of this observation, the syndrome of congestive heart failure has been hypothesized to be due to a deficiency in ANP. The current study was designed to correlate the cardiac content of ANP (determined by immunohistochemical techniques) with plasma circulating ANP (determined by radioimmunoassay). alpha-ANP antibodies were used for both determinations. The content of ANP in the atria was based on the degree of immunoreactive staining present (1 = lowest; 5 = highest), as graded by two observers. The mean granularity score of the cardiomyopathic hamsters was decreased (2.1 +/- 0.3) in comparison with that of age- and sex-matched control animals (3.5 +/- 0.5; P less than 0.05). In contrast, circulating immunoreactive ANP was higher in the hamsters with congestive heart failure than in the control animals--185.5 +/- 27.2 pg/ml versus 77.7 +/- 10.8 pg/ml (P less than 0.005). This study demonstrates that an inverse relationship exists between ANP content in the atria and circulating ANP. Furthermore, this study suggests that these hamsters with congestive heart failure are not deficient in ANP; rather, secretion of ANP is stimulated and storage of the peptide, represented by atrial granularity, is reduced.     

Increase in circulating levels of cardiac natriuretic peptides after hormone replacement therapy in postmenopausal women.

The mechanisms that mediate the cardioprotective action of steroid hormones in postmenopausal women are poorly understood. To study the inter-relationship between female steroid hormones and cardiac natriuretic peptides, plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured in postmenopausal women, both before and after oestrogen replacement therapy. A total of 22 healthy postmenopausal women (mean age 51.9+/-4.6 years) were enrolled in the study; all had been postmenopausal for at least 1 year and all reported climacteric symptoms accompanied by increased levels of follicle-stimulating hormone (>30 m-i.u./ml) and luteinizing hormone (>20 m-i.u./ml), and a reduction in oestradiol (<25 pg/ml). All women were given hormone replacement therapy with transdermal oestradiol, either patch (50 microg/24 h) or gel (1 mg/day), cyclically combined with oral dihydrogesterone (10 mg/day for 12 days/month, on days 19-30 of the month). ANP and BNP were measured directly in plasma samples with specific and sensitive immunoradiometric assays before and after hormone replacement therapy (transdermal oestradiol combined with oral dihydrogesterone). Body weight, arterial blood pressure and echocardiographic examination values did not change after hormone replacement therapy. As expected, serum oestradiol increased significantly and gonadotropins decreased as an effect of the hormone replacement therapy. On average, both ANP and BNP had increased significantly after 3 months of hormone replacement therapy [ANP: before treatment, 17.6+/-9.6 pg/ml; after, 23.6+/-5.6 pg/ml (P=0.0173); BNP: before treatment, 12.6+/-10.2 pg/ml; after, 19.8+/-14.0 pg/ml (P<0.0001)]. Our study indicates that hormone replacement therapy for a period of 3 months induces a rise in the circulating levels of cardiac natriuretic hormones in postmenopausal women. Our data also suggest the working hypothesis that cardiac natriuretic peptides may play an important role in mediating the cardioprotective effects of female steroid sex hormones in women throughout life.     

Measurement of low angiotensin concentrations after ethanol and Dowex extraction procedures

Current radioimmunoassays do not demonstrate total absence of angiotensin II during converting enzyme inhibition. To assess the meaning of plasma angiotensin II determinations during converting enzyme inhibition, plasma angiotensin I and II levels of normotensive humans during maximal converting enzyme inhibition by single oral doses of CGS 13945, MK 421, or MK 521 were compared with those of anephric rats (18 hr after nephrectomy) after intravenous administration of MK 422 (1 mg/kg). Prior to radioimmunoassay, plasma was extracted with Dowex for angiotensin II and blood extracted with ethanol for angiotensin I. During converting enzyme inhibition, in the 20 normotensive subjects plasma angiotensin II was 6.3 +/- 2.3 pg/ml (mean +/- S.D.) and blood angiotensin I was 65 +/- 59 pg/ml. In the nephrectomized rats, plasma angiotensin II was 8.9 +/- 2.3 pg/ml without converting enzyme inhibitor (n = 15) and 7.6 +/- 2.8 with MK 422 (n = 14), and blood angiotensin I was 9.8 +/- 2.4 pg/ml and 8.2 +/- 0.7, respectively. Dowex extraction of Tris buffer containing no angiotensin II provided blank values ranging from 5.0 to 7.8 pg/ml (n = 5). Thus plasma angiotensin II of normotensive humans treated with converting enzyme inhibitors fell to blank levels even in the presence of markedly elevated plasma angiotensin I. Angiotensin II concentrations in anephric rats with or without converting enzyme inhibition were the same. We therefore conclude that plasma levels of angiotensin II below 8 pg/ml measured after Dowex extraction probably reflect complete converting enzyme inhibition and virtual absence of angiotensin II generation.     

A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma, using a tracer with an immobilized glycouril agent

A highly specific and sensitive radioimmunoassay (RIA) for alpha-human atrial natriuretic peptide (hANP[1-28]) in plasma was developed. The assay used a [125I]monoiodotyrosyl-hANP[1-28] tracer, prepared with an immobilized glycouril agent (Protag) and purified by high pressure liquid chromatography (HPLC), and a highly specific antiserum raised against hANP[1-28], coupled to keyhole limpet haemocyanin, in sheep. Plasma was extracted using C-18 Seppak cartridges. A good parallelism was found after dilution prior to extraction of plasma of patients with congestive heart failure (CHF) or of plasma of healthy subjects. Recovery of hANP[1-28] added to plasma was 96%. The limit of detection was 0.8 pg/tube, intra- and inter-assay variation were 9 and 12%, respectively. Mean plasma ANP values in 25 normal persons with a normal salt intake was 26.0 +/- 15.5 (+/- SD) pg/ml. Plasma levels of 18 subjects (7 normals, 11 CHF) were measured using four different antisera after the extraction step. High correlations were found between the values obtained with these four antisera.     

Cardiac secretion and renal clearance of atrial natriuretic peptide in normal man: effect of salt restriction

1. Previous studies of endogenous atrial natriuretic peptide (ANP) in humans have examined changes in plasma levels, rather than regional secretion and clearance of the peptide. Using arterial and selective venous catheterization and sampling, and measurement of regional organ flow, we measured haemodynamics, cardiac secretion of ANP and renal clearance of ANP in six healthy volunteers at rest, on a normal sodium diet. 2. Salt restriction decreases plasma concentrations of ANP. We assessed the contribution of the heart and kidney to this decrease, by measuring cardiac secretion and renal clearance of ANP at the termination of a low salt diet. 3. Twenty-four hour urinary sodium excretion fell on the low salt diet from 163 to 29 mmol/day [standard error of the difference (SED)+/- 14, P less than 0.001]. Body weight decreased on salt restriction from 76.4 to 75.4 kg (SED +/- 0.33, P less than 0.05). Brachial mean arterial pressure fell by 6% (P less than 0.05), but right atrial pressure was unchanged. Renal vein plasma renin activity increased by 56% with sodium restriction (P less than 0.01), whereas arterial ANP concentrations fell by 39% (P less than 0.05). 4. Coronary sinus ANP levels fell from 417 to 268 pg/ml (SED +/- 74, P less than 0.05), whereas renal vein concentrations were unaltered. There was a 47% decrease in cardiac secretion of ANP in the low salt state (P less than 0.05). Net extraction of ANP across the kidney (about two-thirds) and renal clearance of ANP were unchanged on the low salt diet. Thus decreased plasma ANP with sodium restriction is due to reduced cardiac secretion.     

Elevated plasma atrial natriuretic peptide levels in diabetic rats. Potential mediator of hyperfiltration.

Infusion of atrial natriuretic peptide (ANP) increases the glomerular filtration rate (GFR), and ANP is released from cardiac myocytes in response to extracellular fluid volume expansion. Since diabetes mellitus is associated with glomerular hyperfiltration and volume expansion, we investigated the relationship between ANP and GFR in diabetic rats given insulin to achieve stable moderate hyperglycemia or normoglycemia. At 2 wk after induction of diabetes, moderately hyperglycemic diabetic rats exhibited elevations of plasma ANP levels averaging 281 +/- 28 pg/ml vs. 158 +/- 15 pg/ml in normoglycemic diabetic rats. In hyperglycemic rats, the GFR was also elevated on average to 2.24 +/- 0.28 ml/min as compared with 1.71 +/- 0.13 ml/min in normoglycemic diabetic rats. To test further the relationship between ANP and GFR in diabetes, moderately hyperglycemic diabetic rats were infused either with a specific ANP antiserum or with nonimmune serum. The infusion of specific ANP antiserum uniformly reduced the GFR on average from 2.38 +/- 0.1 ml/min to 1.60 +/- 0.1 ml/min, whereas the infusion of nonimmune serum was without effect. It is concluded that elevated endogenous ANP levels contribute to the hyperfiltration observed in early diabetes in the rat.     

Effect of insulin on motilin release in man

The effect of insulin on motilin release was investigated by use of the euglycemic glucose clamp technique. By use of this technique plasma glucose concentration was maintained constant at 80-90 mg/100 ml, and plasma insulin immunoreactivity (IRI) was increased from 15 +/- 6 microU/ml to 171 +/- 22 microU/ml in 10 min, and remained at this level for 2 hr. Plasma motilin like immunoreactivity (MLI) concentration decreased within 10 min from 199 +/- 36 pg/ml to 120 +/- 28 pg/ml and remained low during the course of study. A significant negative correlation between MLI and IRI concentrations (r = -0.72, p less than 0.01) was observed. The present results indicate that the suppressive effect of insulin on motilin release is a direct action of insulin and is not mediated by glucose.