A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process such as Sep-Pak C-18 cartridge treatment. An antiserum was prepared from rabbits immunized with alpha-human ANP (alpha-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.3 pg/tube of synthetic alpha-hANP utilized as authentic standard. Recovery of alpha-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 healthy subjects, the plasma ANP-concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.     

Atrial natriuretic peptide and vasopressin in human plasma

Using a specific radioimmunoassay for atrial natriuretic peptide (ANP), plasma immunoreactive ANP was measured in 17 normal subjects and 83 patients with various diseases. Plasma ANP concentration in normal subjects was 14.1 +/- 1.7 pg/ml (mean +/- S.E.). Relatively high plasma ANP concentrations were detected in patients with diabetes mellitus, hyperthyroidism, atrial fibrillation and liver cirrhosis. Plasma ANP concentrations in the patients correlated positively with mean arterial blood pressure and plasma AVP concentrations. Plasma ANP concentrations in the patients also had positive correlations with left atrial dimension and left ventricular diastolic dimension determined by echocardiography. Another positive correlation was observed in the patients between plasma AVP concentrations and mean arterial blood pressure. These results suggest that ANP is a volume regulatory hormone but also that ANP may be involved in the blood pressure regulating system.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Atrial natriuretic peptide during water deprivation or hemorrhage in rats. Relationship with arginine vasopressin and osmolarity.

The concentrations of atrial natriuretic peptide (ANP) in atria, hypothalami and plasma were investigated in relation to the variations of the plasma endogenous immunoreactive arginine vasopressin (Ir-AVP) during water deprivation or hemorrhage in normal conscious Wistar rats. Furthermore, the in vitro and in vivo effect of extracellular hyperosmolarity on ANP release from right atrium and hypothalamus was examined. Water deprivation elevated circulating immunoreactive ANP (Ir-ANP: pg/ml) to 153 +/- 7 (24 h); 174 +/- 1 (48 h) from the control level (109.6 +/- 7.8). This increase in Ir-ANP concentration which correlated with atrial (r = -0.93) or hypothalamic (r = -0.87) Ir-ANP content decrease, was associated with significantly enhanced levels of plasma Ir-AVP, plasma sodium, osmolarity and hematocrit. An acute volume depletion by hemorrhage significantly reduced plasma Ir-ANP (67 +/- 8.4 pg/ml) from the sham operated level (140 +/- 18 pg/ml). Plasma Ir-AVP was elevated dramatically (207.4 +/- 53.4 pg/ml) compared with the sham operated level (8.8 +/- 2.6 pg/ml). These results, indicating the lack of correlation between plasma Ir-ANP and Ir-AVP in vivo, suggest that the ANP secretion, which is regulated mainly by plasma volume, may be modulated by a change in plasma osmolarity. Extracellular hyperosmolarity stimulated the ANP release from superfused sliced normal rat atria and hypothalami.     

Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation

A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction.     

Decreased content in left atrium and increased plasma concentration of atrial natriuretic polypeptide in spontaneously hypertensive rats (SHR) and SHR stroke-prone

The atrial contents and concentrations, and the plasma concentrations of atrial natriuretic polypeptide (ANP) in spontaneously hypertensive rats (SHR) and SHR stroke-prone (SHRSP) were measured and compared with those of age-matched Wistar Kyoto rats (WKY) using a specific radioimmunoassay (RIA) for alpha-rat ANP (alpha-rANP). The contents of alpha-rANP-LI in the atria of SHR (19.0 +/- 0.9 micrograms, mean +/- SEM) and SHRSP (19.3 +/- 0.6 micrograms) were significantly lower than that of WKY (22.8 +/- 1.4 micrograms) (p less than 0.05). The atrial concentration of alpha-rANP-LI was also significantly lower in SHR (248.2 +/- 11.3 ng/mg, p less than 0.05) and tended to be lower in SHRSP (272.2 +/- 12.4 ng/mg) than that of WKY (300.0 +/- 14.2 ng/mg). Furthermore, the concentrations in the left auricles of SHR and SHRSP were significantly lower than that of WKY (p less than 0.01 and p less than 0.05, respectively). In contrast, no significant difference was observed in the alpha-rANP-LI concentrations in the right auricles of WKY, SHR and SHRSP. Gel filtration studies coupled with RIA showed that gel filtration profiles of the extracts from the right and left auricles of WKY, SHR and SHRSP were essentially identical. The plasma alpha-rANP-LI levels in SHR (260 +/- 34 pg/ml) and SHRSP (319 +/- 19 pg/ml) were significantly higher than that in WKY (170 +/- 17 pg/ml) (p less than 0.05 and p less than 0.01, respectively). These results suggest that the secretion of ANP from the heart is increased in SHR and SHRSP compared with WKY.     

Brain natriuretic peptide is a novel cardiac hormone

Using a radioimmunoassay for brain natriuretic peptide (BNP), we have measured levels of BNP-like immunoreactivity (-LI) in extract of the porcine heart, in perfusate from the isolated porcine heart and in porcine plasma. BNP-LI was detected in the extract of the atrium, though no detectable amount of BNP-LI (more than 1 ng/g) was present in the ventricle. The BNP-LI level in the porcine atrium was 148.7 +/- 23.3 ng/g. BNP-LI was also detected in the perfusate from the heart. Basal secretory rate of BNP was 3.18 +/- 0.76 ng/min. Moreover, BNP-LI was detected in porcine plasma at the concentration of 4.2 +/- 1.3 pg/ml. Gel filtration studies showed that BNP is present in the atrium as a large molecule and is secreted into the circulation as a small molecule. The percentage of BNP-LI to atrial natriuretic peptide (ANP)-LI was almost the same among the extract, the perfusate and the plasma (2-3 percent). These results indicate that BNP is synthesized in and is secreted into the circulation from the heat in a similar fashion as ANP.     

Effects of ovine corticotropin-releasing hormone and FK 33-824 (met-enkephalin analogue) on the secretions of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in patients with Addison's disease

The responses of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH) and IR-ACTH levels to ovine corticotropin-releasing hormone (CRF) and FK 33-824 (Met-Enkephalin analogue) were studied in nine patients with Addison's disease. The basal plasma levels (mean +/- SE) of IR-Nt, IR-Ep, IR-LPH and IR-ACTH were significantly higher in patients with Addison's disease (4459 +/- 975 pg/ml, 132 +/- 25 pg/ml, 4425 +/- 1030 pg/ml, 553 +/- 89 pg/ml, respectively) than in the normal controls (202 +/- 38 pg/ml, 7 +/- 2 pg/ml, 101 +/- 18 pfi/ml, 53 +/- 16 pg/ml, respectively). Ovine CRF produced rapid and concomitant increases in plasma levels of IR-Nt, IR-Ep, IR-LPH and IR-ACTH. Ep and ACTH levels reached a peak at 30 min. On the other hand, Nt and LPH levels reached a peak at 60 min and these levels gradually decreased up to 120 min. The molar concentrations of these IR-peptides in plasma were changed in close parallel fashion to one another. FK 33-824 produced a pronounced and concomitant fall in IR-Nt, IR-EP, IR-LPH, and IR-ACTH levels. These results support the theory that Nt, Ep, LPH and ACTH are produced simultaneously from POMC as a common precursor in the pituitary gland and are secreted concomitantly under various conditions such as stimulation by CRF and inhibition by FK 33-824 in patients with Addison's disease.     

Radioimmunoassay for insulin-like growth factor I (IGF-I) using biosynthetic IGF-I

We produced antiserum to insulin-like growth factor I (IGF-I), and developed a specific and sensitive radioimmunoassay (RIA) for IGF-I using the biosynthetic IGF-I. This antiserum to IGF-I was specific for IGF-I; no cross-reactivities with multiplication stimulating activity, porcine insulin or human growth hormone (hGH) were detected. The sensitivity was 10-25 pg/tube with 50% displacement at 125 pg/tube. The intra- and inter-assay coefficients of variation for IGF-I were 5.4 and 9.7%, respectively. The plasma IGF-I levels as determined by RIA in normal adults (N = 46), patients with active acromegaly (N = 31), and pituitary dwarfs (N = 31) were 21.6 +/- 1.0, 157.3 +/- 17.0, and 2.5 +/- 0.3 ng/ml (Mean +/- SEM), respectively, indicating the levels were GH-dependent. The plasma IGF-I levels were significantly increased from 2.2 +/- 0.2 to 26.5 +/- 3.2 ng/ml after hGH administrations for three consecutive days in five pituitary dwarfs. The IGF-I levels were low in patients with hypothyroidism and liver cirrhosis, but were normal in patients with chronic renal failure. These data confirm previous reports and this radioimmunoassay proves useful in evaluating plasma IGF-I levels.     

Raised plasma endothelin-I concentration following cold pressor test

Plasma concentration of immunoreactive endothelin-1 was measured by radioimmunoassay in 6 healthy subjects before and following cold pressor test by immersion of one fore-arm into ice-water. Mean (SEM) plasma endothelin-1 concentration rose from 1.2 (0.7) to peak value 8.4 (2.3) pg/ml in venous plasma from the immersed hand, and, reaching peak 2 minutes later, from 1.4 (0.5) to 4.6 (2.3) pg/ml in venous plasma from the contralateral hand. In 66 healthy control subjects, venous plasma concentration of endothelin-1 was 2.9 +/- 1.2 pg/ml (mean +/- SD). Exposure to cold is associated with raised blood levels of endothelin-1, which points to a relation between endothelin-1 and vasoconstriction associated with low temperature.     

The ratio of plasma bioactive to immunoreactive ACTH-like activity increases with gestational age in the fetal lamb.

The fetal ovine pituitary-adrenal axis plays an important role in the timing of parturition, in fetal lung maturation, and in fetal and neonatal responses to stress. While the ovine pituitary during the last third of gestation (term = 145 days) is capable of secreting immunoreactive ACTH (iACTH) in response to various stimuli, plasma cortisol levels frequently do not reflect the rise in plasma ACTH. Therefore, we examined the relationship between plasma iACTH and steroidogenic ACTH-like activity (bACTH) in a group of immature fetal lambs (Group I: gestational age = 97 +/- 2 days, mean +/- SEM, n = 16) and a group of near-term fetuses (Group II: gestational age = 136 +/- 1 days, n = 13) following acute exteriorization. Plasma iACTH was determined by RIA. Plasma bACTH was determined by the ability of glass-extracted material to stimulate corticosterone (B) production in an acutely dispersed rat adrenal bioassay. Plasma iACTH and bACTH levels varied among animals within age groups, with iACTH tending to be higher in immature fetal lambs (Group I) than near-term lambs (Group II) and bACTH being higher (P < 0.05) near term than earlier (Group I: iACTH = 807 +/- 273 pg/ml, bACTH = 173 +/- 44 pg/ml; Group II: iACTH = 405 +/- 85 pg/ml, bACTH = 371 +/- 96 pg/ml). The proportion of iACTH that had biologic activity (e.g. B/I ratio) was significantly greater in the older than in the younger fetuses (Group II: B/I = 0.862 +/- 0.109; Group I: B/I = 0.462 +/- 0.105 P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor in human plasma

A reproducible and sensitive radioimmunoassay (RIA) was developed to measure ANF in human plasma. Immunoreactive ANF was extracted from plasma with Sep-Pak cartridges, using 0.2% ammonium acetate (pH 4) with acetonitrile. The sensitivity of the assay was 3.9 pg/ml. The coefficient of variance for inter-assay and intra-assay was 16.8% and 6.8%, respectively. In normal healthy subjects (n = 67), ANF content was 11.9 +/- 1.3 pg/ml (mean +/- SEM). Significantly-higher ANF concentrations were found in proximal coronary sinus blood, being 6 to 37 times greater than in the peripheral circulation. Comparison of the prior extraction method with direct RIA revealed a good correlation (r = 91) in samples containing higher than 100 pg/ml ANF. No correlation was observed with lower values. The elution profiles of reverse-phase HPLC of peripheral and coronary sinus plasma extracts were similar but somewhat complex, with the main immunoreactive peak corresponding to a low-molecular-weight peptide.     

Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma

A direct radioimmunoassay for ANF in human plasma was developed. A synthetic alpha-human atrial peptide (Ser 99-Tyr 126) was used for preparation of the iodinated tracer and the standards. The sensitivity of the method is 1.9 pg/ml. Concentration of immunoreactive ANF (IR-ANF) in plasma of 59 clinically normal subjects was 65.3 +/- 2.5 pg/ml (mean +/- SE). In two patients who underwent atrial pacing an increase of about 100 percent in circulating IR-ANF was observed. IR-ANF was extracted from human plasma by Vycor glass and purified by HPLC. The main immunoreactive isolated peak contained a low molecular weight peptide.     

Plasma levels of 7B2 (a novel pituitary polypeptide) and its molecular forms in plasma and urine in patients with chronic renal failure: possible degradation by the kidney

Plasma immunoreactive (IR)-7B2 was measured in patients with chronic renal failure (CRF), using a specific radioimmunoassay. The mean (+/- S.E.M.) concentration of plasma IR-7B2 in CRF patients under hemodialysis (502 +/- 36 pg/ml, n = 27) was significantly higher than that in normal subjects (men, 52.9 +/- 1.7 pg/ml (n = 179); women, 55.8 +/- 1.3 pg/ml (n = 198]. Significant correlations between plasma levels of IR-7B2 and those of blood urea nitrogen, creatinine and beta 2-microglobulin were evident in non-dialyzed CRF patients. In the analyses of pooled plasma and urine obtained from normal subjects on gel permeation chromatography, a major peak of IR-7B2 was observed at an apparent molecular weight of 20,000 in the plasma, and at a position of a smaller molecular weight in the urine. These results suggest that 7B2 is degraded mainly in the kidney and that measurement of plasma 7B2 may serve as an appropriate tool for assessing renal function.     

Locally different role of atrial natriuretic peptide (ANP) in the pericardial fluid

Pericardial fluid (PF) contains several vasoactive agents in higher concentrations than venous plasma (VP). However, with human atrial natriuretic peptide (ANP) controversial data have been reported in earlier studies performed on a limited number of patients (less than 20). The present study was designed to characterize the ANP levels in human PF and cardiac tissues, and to ascertain whether myocardial ischemic state is a major factor in determining ANP production of the human heart. In a total of 316 consecutive patients undergoing open heart surgery ANP levels in VP, PF, atrial and ventricular tissues were measured by radioimmunoassay and analyzed by high-performance liquid chromatography (HPLC). The data are presented as median and 25th-75th percentiles. Our results showed ANP concentration [ANP] of PF significantly exceeded that of VP and [ANP] in the atrial tissue was significantly higher than in the ventricular tissue (p < 0.001). In patients without myocardial ischemia (valvular heart disease) [ANP] in the PF was 258.3 (189.9-342.5) pg/ml, in the VP 28.4 (11.7-57.6) pg/ml and 151.7 (78.4-447.6) ng/mg in the atrial, 0.4 (0.2-1.6) ng/mg in the ventricular tissue. The corresponding values for patients with coronary artery disease were 208.1 (153.8-318.9) pg/ml in the PF, 19.8 (9.4-27.9) pg/ml in the VP, 129.6 (66.5-455.0) ng/mg in the atrial and 1.0 (0.1-1.8) ng/mg in the ventricular tissue. The ventricular tissue levels correlated to the atrial tissue levels (r = 0.317; p < 0.05). Great difference (p < 0.001) was found in the atrial tissue levels between females [414.6 (119.7-734.4) ng/mg] and males [105.4 (65.3-204.2) ng/mg]. In HPLC analysis the majority of the pericardial fluid and tissue ir-ANP coeluted with human ANP [99-126]. In conclusion, [ANP] in PF of cardiosurgical patients is higher by an order of magnitude than in VP. Intrapericardial ANP may reflect the peptide concentration in the myocardial interstitium and may represent a paracrine regulatory mechanism, which seems independent of ANP-induced putative antiischemic influences.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Radioimmunoassay of secretin in plasma.

A sensitive, specific and reproducible radioimmunoassay for secretin is described. Antibodies were readily produced against low microgram quantities of synthetic secretin. The secretin antibodies did not cross-react with the structurally similar G.I.P., V.I.P., or glucagon. Synthetic secretin was iodinated using Chloramine "T" and purified by a two-state procedure incorporating gel filtration and radient elution from a cation exchange column. Plasma samples were found to produce variable interference in the assay necessitating the incorporation of secretin-free "blands" for each patient's plasma. Production of secretin-free plasma was by incubation of plasma samples at 37 degrees C for 96 hours. The sensitivity of the assay was 12.5-25 pg/ml. Normal fasting secretin levels were 21 +/- S.E. 7 pg/ml. A mean rise in plasma secretin to 220 pg/ml was observed after intraduodenal acidification.     

Development of a sensitive somatostatin radioimmunoassay and its application to plasma of stressed and non-stressed rats

A sensitive somatostatin radioimmunoassay was developed and immunoreactive somatostatin was measured in plasma of different vessels of stressed and non-stressed rats. Detection limit of the assay is 4 pg/ml. Serial dilutions of rat plasma run parallel to the standard curve. On gel chromatography of rat plasma immunoreactive somatostatin elutes at the position of synthetic somatostatin. Immunoreactive somatostatin in plasma of non-stressed rats is (mean +/- 1 SEM) 369 +/- 58, 244 +/- 66, 273 +/- 61, 260 +/- 44, 359 +/- 80 pg/ml in aorta abdominalis, vena cava, vena renalis, vena jugularis, vena porta respectively. After stress mean immunoreactive somatostatin was higher in plasma of all vessels, in aorta abdominalis (p less than 0.01), vena renalis and vena porta about 50 per cent, in vena jugularis and vena cava about 90 per cent (p less than 0.002). We conclude that under conditions like stress somatostatin circulates in increased amounts and perhaps reaches organs distant from documented sources.     

High plasma cholecystokinin response following ingestion of test meal by patients with non-insulin dependent diabetes mellitus

Postprandial responses of plasma cholecystokinin (CCK) in patients with non-insulin dependent diabetes mellitus (NIDDM) were studied with a CCK specific radioimmunoassay. After the ingestion of a liquid test meal, plasma CCK levels increased from the basal level of 9.8 +/- 1.1 pg/ml to a peak of 19.4 +/- 1.8 pg/ml at 20 min in healthy subjects (n = 10). The ingestion of a test meal in patients with NIDDM (n = 10) resulted in a significantly greater increase of plasma CCK than in healthy subjects and a significant increase of plasma CCK from a basal level of 14.2 +/- 4.4 pg/ml to a peak of 47.4 +/- 12.4 pg/ml at 10 min.     

Presence of the atrial natriuretic peptide in human cerebrospinal fluid

Using a highly sensitive and specific radioimmunoassay (RIA) for detection of the atrial natriuretic peptide (ANP), the presence of alpha-human ANP (alpha-hANP) in human cerebrospinal fluid (CSF) was confirmed. Its concentration in CSF, 3.6 +/- 2.3 pg/ml, n = 16, mean +/- SD, was remarkably lower than that in the plasma (161.8 +/- 157.4, p less than 0.0001). The regression coefficient between these concentrations was 0.320 (p = ns). Gel permeation chromatography conducted in conjunction with RIA indicated ANP in CSF to be eluted at the position of a low molecular weight form corresponding to alpha-hANP. No high molecular weight form could be detected. But in the plasma, both low and high molecular forms were found to be present. It is thus evident that ANP is present in human CSF and its origin may possibly be the brain and not the atrium.