Determination Of Plasma Renin Activity Research Articles

Stability of direct renin concentration and plasma renin activity in EDTA whole blood and plasma at ambient and refrigerated temperatures from 0 to 72 hours.

The aim of this study was to determine the appropriate transport and storage conditions for blood taken for direct renin concentration and plasma renin activity measurement, and whether cryoactivation of prorenin is seen at time points relevant to clinical practice. Blood was extracted from n=10 volunteers into K2-EDTA tubes. Stability of renin was assessed in whole blood stored at room temperature (15-25°C) and in the refrigerator (2-8°C) at 0h, 8h, and 24h. The stability of renin in plasma was determined under the same conditions at 0h, 24 h and 72h. Stability of plasma renin activity and direct renin concentration in whole blood stored at room temperature was found to be acceptable for up to 24h. At refrigerated temperature, whole blood stability was acceptable for measurement of direct renin concentration up to 8h and plasma renin activity up to 24h. In contrast, plasma renin activity was not stable in plasma stored at either room or refrigerated temperatures up to 24h; however, direct renin concentration had acceptable stability in plasma stored at room temperature for up to 24h, but stability was unacceptable at refrigerated temperatures. Samples collected for plasma renin activity and direct renin concentration should be transported as whole blood to optimise stability. After sample processing, plasma can be kept at room temperature for up to 24h for direct renin concentration, however, for determination of plasma renin activity separated plasma should be analysed or frozen as soon as possible.

Quantitation of Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartters and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semiautomated and simple method for the accurate and precise measurement of PRA from 500μL of plasma (250μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3h AngI generation step at 37°C in buffering conditions at pH 6, the reaction is quenched with 10% formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0ng/mL, which corresponds to PRA values of 0.11-10.0ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8-5.0% for BioRad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43ng/mL/h. At AngI concentrations of 0.06ng/L (corresponding to a PRA of 0.02ng/mL/h), signal-to-noise ratio is 50:1, indicating that the limit of quantitation is well below the level required for clinical use.

A quality control system for ligand-binding assay of plasma renin activity: Proof-of-concept within a pharmacodynamic study

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded “Labeling of Enalapril in Neonates up to Adolescents” project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

Quantitation of Plasma Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartter and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semi-automated and simple method for the accurate and precise measurement of PRA from 500 μL of plasma (250 μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10 % formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0 ng/mL which corresponds to PRA values of 0.11-10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8 to 5.0 % for Bio-Rad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal to noise ratios are 50:1 indicating that the limit of quantitation is well below the level required for clinical use.

A previous history of repeated amphetamine exposure modifies brain angiotensin II AT1 receptor functionality

Previous results from our laboratory showed that angiotensin II AT1 receptors (AT1-R) are involved in the neuroadaptative changes induced by amphetamine. The aim of the present work was to study functional and neurochemical responses to angiotensin II (ANG II) mediated by AT1-R activation in animals previously exposed to amphetamine. For this purpose male Wistar rats (250–320g) were treated with amphetamine (2.5mg/kg/day intraperitoneal) or saline for 5days and implanted with intracerebroventricular (i.c.v.) cannulae. Seven days after the last amphetamine administration the animals received ANG II (400pmol) i.c.v. One group was tested in a free choice paradigm for sodium (2% NaCl) and water intake and sacrificed for Fos immunoreactivity (Fos-IR) determinations. In a second group of rats, urine and plasma samples were collected for electrolytes and plasma renin activity determination and then they were sacrificed for Fos-IR determination in Oxytocinergic neurons (Fos-OT-IR). Results: Repeated amphetamine exposure (a) prevented the increase in sodium intake and Fos-IR cells in caudate-putamen and accumbens nucleus induced by ANG II i.c.v. (b) potentiated urinary sodium excretion and Fos-OT-IR in hypothalamus and (c) increased the inhibitory response in plasma renin activity, in response to ANG II i.c.v. Our results indicate a possible functional desensitisation of AT1-R in response to ANG II, induced by repeated amphetamine exposure. This functional AT1-R desensitisation allows to unmask the effects of ANG II i.c.v. mediated by oxytocin. We conclude that the long lasting changes in brain AT1-R functionality should be considered among the psychostimulant-induced neuroadaptations.

An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI)

Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC–MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC–MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC–MS/MS. The assay is performed on 350μL of plasma, and has a linear range from 0.08 to 5.3ng/L/s in the reflector mode, and 0.04 to 5.3ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC–MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R2 value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics.

Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension.

Plasma renin activity (PRA) is an essential analytical tool for screening and diagnosis of secondary forms of hypertension. Typically, PRA is measured by competitive radioimmunoassay, but there are significant drawbacks to this technique including non-specificity, long analysis times, narrow calibration range, and the requirement for radionucleotides. In this paper, we report a method for plasma renin activity determination by immuno-MALDI mass spectrometry detection. This method overcomes the issues of non-specificity and long analytical times present with RIA, and does not require the use of radionucleotides. As an initial methodological evaluation, plasma renin activity results obtained by radioimmunoassay, LC/ESI-MS/MS, and immuno-MALDI on 64 samples from an outpatient primary aldosteronism screening program have been compared. A strong correlation was found between immuno-MALDI and radioimmunoassay (R2 = 0.9412, 62/64 within the 95% CI of the Bland-Altman plot), and iMALDI and LC/ESI-MS/MS (R2 = 0.9471, 62/64 within the 95% CI of the Bland-Altman plot). Technical replicates showed a 4.8% CV, while inter- and intra-day replicates showed CVs of 17.3% and 17.2% respectively. We have developed an assay capable of measuring PRA without the use of radionucleotides. This immuno-MALDI approach affords the specificity of MS while avoiding the long analytical run times and technical problems associated with HPLC. With the use of robotic sample preparation to optimize precision, this assay should be adaptable to clinical environments.

Abstract 18811: Plasma Renin Activity Test Versus US Joint National Committee Guidelines for Selecting Initial Antihypertensive Drug

Background: US National Guidelines promote initiating treatment for nearly all hypertensive patients with a natriuretic drug. We now report a double-blind, randomized controlled trial to compare that with a Plasma Renin Activity (PRA) test guided therapeutic approach. Methods: Hypertensive participants (n=164) consented to (i) replacement of antihypertensive drugs with placebo for at least 4 weeks and then (ii) a 5 month double blind study to compare BP control and BP change achieved by initial monotherapy: Experimental = guided by PRA (n=82) v. Control = hydrochlorothiazide (HCTZ) (n=82). Results: Eligible subjects were 45% males with a mean±SD age 60±9, entry BP 152±10/86±10mmHg,and median (IQR) PRA 0.7 (0.2, 1.4)ng/mL/hr. Experimental subjects were given a diuretic if PRA <0.65 (n=32?), or olmesartan if PRA ≥0.65 ng/mL/hr (n=50?). At 4 months, 72% of both Experimental and Control groups achieved BP <140/90mmHg, (132±15/76±11 v. 134±14/79±9mmHg,p=.39/.18 respectively). As expected, in subjects with PRA<0.65ng/mL/hr BP decline was similar in Experimental and Control groups. By contrast, in 91 patients with PRA ≥ .65ng/mL/hr, after adjusting for entry BP Experimental subjects (n=50) had a significantly greater mean±SE fall in systolic BP [_-4.9±2.4mmHg (p=.04)] and a borderline significantly greater fall when not adjusted (_-4.5±2.3mmHg, p=.06). Conclusions: Effectiveness of PRA guided therapy is identical to US National Guidelines for patients with PRA < 0.65 ng/mL/hr. However, it significantly improved BP treatment in patients with PRA ≥ 0.65ng/ml/hr. Moreover, in this clinical trial of moderately hypertensive patients, initial monotherapy lowered BP to <140/90 mmHg in nearly 3/4 of subjects. These results suggest that routine PRA determination can improve both the effectiveness and efficiency of hypertension treatment.

Efficacy of antihypertensive treatment based on plasma renin activity: An open label observational study

Background. We investigated the extent of blood pressure (BP) reduction and control (<140/90 mmHg) in patients treated with appropriate or inappropriate drugs according to their plasma renin activity (PRA) level (natriuretic drugs and renin–angiotensin system blockers for low-renin and high-renin hypertension, respectively). Patients and methods. One hundred and seventy Caucasian untreated hypertensive patients (61 females), aged 18–70 years, participated to the study. Patients with secondary hypertension, diabetes or established cardiovascular or renal disease were excluded. The physician prescribed an antihypertensive monotherapy chosen among all drug classes, unaware of patient's PRA levels. We compared effect of an inappropriate or appropriate drug, evaluating BP values after a month of treatment. Results. Rate of BP control was not significantly higher in patients treated with an appropriate drug than the others (38% vs 29%, p=0.24). However, in a regression analysis, final diastolic BP (DBP) was lower in subjects treated with an appropriate drug (β=−2.84, p=0.03). Conclusions. The present study does not clearly support the use of PRA in a general population of hypertensive patients to optimize BP control. However, the greater efficacy of a drug appropriate to PRA in reducing DBP may be clinically helpful in young hypertensive patients. Future studies are warranted to evaluate if PRA determination enhances the therapeutic success in patients with predominantly high values of DBP.

Towards the Development of an Immuno MALDI (iMALDI) Mass Spectrometry Assay for the Diagnosis of Hypertension

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in the regulation of plasma volume and arterial blood pressure. One of the most common diseases of the RAAS is the autonomous production of aldosterone by the adrenal glands, caused by either bilateral adrenal hyperplasia or an aldosterone-producing adenoma. This condition, known as primary aldosteronism, is a treatable and often curable form of hypertension. The measurement of plasma renin activity (PRA), as determined by radioimmunoassay for angiotensin I is essential to the diagnosis of primary aldosteronism. However, accurate determination of PRA is often hampered by low plasma concentrations of angiotensin I. Here, we report the use of immuno-MALDI (iMALDI) as a highly sensitive and specific method for the absolute quantitation of angiotensin I in plasma. iMALDI permits concentration determination by affinity-capture of angiotensin I and a stable-isotopically labeled standard (SIS) peptide on immobilized anti-peptide antibodies. The affinity beads are placed on the MALDI target, permitting automated analysis of large numbers of patient samples. Pretreatment of the plasma is not required, and this method is suitable for the accurate determination of angiotensin I in whole plasma. The calibration curve generated using this method was linear over a 50-fold concentration range in plasma, with a correlation coefficient of 0.984. MS/MS sequence confirmation provides absolute specificity. The iMALDI angiotensin I assay, therefore, has the potential to be developed into a method for determining PRA that has advantages in time, in specificity, and in safety.

Pre-analytical and analytical considerations for the determination of plasma renin activity

Background Primary hyperaldosteronism diagnosis is helped by assaying renin concentrations either by immunodetection (active renin) or by enzymatic reaction followed by angiotensin I immunoassay (plasma renin activity). We investigated the impact of pre-analytical and analytical conditions on a PRA assay. Methods PRA was assayed using a commercial kit. Firstly, a retrospective analysis of PRA results from 1 yr was performed. Secondly, the impact of pre-analytical temperature conveyance/storage of samples was tested (4 and 25 °C). Thirdly, 2 durations of enzymatic reaction 1.5 and 18 h were tested. Results Retrospectively, 9.5% samples displayed elevated “blanks”: calculating PRA with or without “blank” subtraction elicited a difference of >10% in 1/3 samples. There was a significant decrease of PRA values in samples left at 25 °C vs 4 °C (50% “blank” increase). Finally, the longer the enzymatic reaction (1.5 vs 18 h), the higher the production of angiotensin I and the better the PRA assay sensitivity. Conclusion We propose a 2 step procedure: a brief 1.5 h enzymatic reaction time followed by a prolonged reaction (18 h) only if PRA is <0.2 ng mL − 1 h − 1 . We would recommend performing “blank” samples for the brief enzymatic reaction time. Finally, conveyance/storage temperature does modify PRA results albeit in our hands this had little clinical impact.

Aliskiren Fails to Lower Blood Pressure in Patients Who Have Either Low PRA Levels or Whose PRA Falls Insufficiently or Reactively Rises

Suppressed baseline plasma renin activity (PRA) levels or large reactive increases in renin secretion are two possible reasons for treatment failure with antirenin system drugs. To investigate their prevalence we reanalyzed data from three published clinical trials of the renin inhibitor aliskiren. Aliskiren failed to lower systolic blood pressure (SBP) by at least 10 mm Hg in half of all patients. It was very effective in two-thirds of medium- to high-renin patients (-19 mm Hg). But BP did not fall in most low-renin patients, or in 30% of medium- to high-renin patients. BP actually rose by >10 mm Hg in 5% of patients taking aliskiren and in >10% of patients when aliskiren was added to an angiotensin receptor blocker (ARB) or angiotensin converting enzyme inhibitor (ACEI). PRA changed in parallel with BP. Although PRA fell in most patients, it actually rose in 5% and in up to 30% when aliskiren was added to an ARB or ACEI. There are two reasons for treatment failure with aliskiren. Many hypertensive patients have large BP falls. But, BP does not fall in most low-renin patients or in medium- to high-renin patients whose PRA levels do not fall sufficiently. If the concept of that treatment resistance is caused by excessive reactive increases in renin secretion is confirmed, then a PRA determination during treatment could be used to guide subsequent addition or subtraction of either natriuretic or antirenin drug types, to thereby correct BP and reduce cardiovascular risk.

11.13 Utility of Determination of Plasma Renin Activity in Choosing the Antihypertensive Treatment

11.13 Utility of Determination of Plasma Renin Activity in Choosing the Antihypertensive Treatment

Is Renin-Based Treatment a Reasonable Strategy to Treat Essential Hypertension?

Plasma renin activity (PRA) levels have been used to define two categories of essential hypertensive patients differing for the basic pathophysiological mechanisms sustaining high blood pressure and for the responsiveness to different antihypertensive agents. In particular, patients with low-renin hypertension should respond better to antisodium-volume drugs and patients with normal- to high-renin hypertension should respond better to renin angiotensin aldosterone system-targeted drugs. Although the experimental verification of this treatment algorithm in both retrospective and prospective analyses of several clinical trials has led to mixed results, the recommendations from the British Hypertension Society can be interpreted in the light of this approach by substituting the direct determination of PRA with the use of demographic parameters (such as race and age), which have been demonstrated to be strictly correlated to PRA levels. Furthermore, the use of PRA screening has also been advocated in cases of resistant hypertension.

Prevalence of Primary Aldosteronism among Unselected Hypertensive Patients: A Prospective Study Based on the Use of an Aldosterone/Renin Ratio above 25 as a Screening Test

Primary aldosteronism (PA) has been considered a rare cause of hypertension. The introduction of the aldosterone/renin ratio (ARR) as a screening test has led to an increase in the detection rate. The aim of this study was to evaluate the prevalence of PA among unselected hypertensive patients by using an ARR >25 as a screening test. We studied 3,000 consecutive unselected hypertensive patients. Blood samples for the determination of plasma renin activity (PRA), aldosterone (ALD) and electrolytes were drawn in the morning, and patients with an ARR >25 underwent intravenous saline infusion as a confirmatory test. Adrenal CT and a dexamethasone suppression test were performed in patients with confirmed PA. Patients with a positive dexamethasone test underwent genetic testing for glucocorticoid-remediable aldosteronism (GRA). Out of 3,000 hypertensives, 684 (22.8%) showed an ARR >25 and 177 of them (5.9% of the whole population) had a positive saline loading test. Only 44 of them (24.8%) were hypokalemic. CT was performed in all the patients with confirmed PA and 53 of them (29.9%) had a solitary adrenal macroadenoma, 112 (63.3%) had bilateral adrenal enlargement and 12 (6.8%) had normal appearing adrenal glands. Of 177 patients given dexamethasone to identify GRA, 8 (4.5%) showed aldosterone suppression but only one (0.1%) tested positive for the chimeric gene. In conclusion, our findings indicate that standardized application of an ARR >25 to unselected hypertensive patients, followed by i.v. saline loading as a confirmatory test, can result in the detection of a large number of patients with PA (5.9% of the studied population), most of whom are normokalemic. Bilateral adrenal hypertrophy represents the more common form of PA.

Neurohormonal, hemodynamic, and electrocardiographic evaluations of healthy dogs receiving long-term administration of doxorubicin

To evaluate diagnostic testing that could be used to establish an early diagnosis of cardiotoxicosis induced by long-term administration of doxorubicin. 13 adult mixed-breed dogs. Procedures-7 dogs were administered doxorubicin chloride (30 mg/m(2), IV, q 21 d for 168 days [cumulative dose, 240 mg/m(2)]), and 6 dogs received saline (0.9% NaCl) solution (5 mL, IV, q 21 d for 168 days; control group). Echocardiography, ECG, arterial blood pressure, plasma renin activity (PRA), and plasma concentrations of norepinephrine and brain natriuretic peptide (BNP) were assessed before each subsequent administration of doxorubicin and saline solution. Dogs that received doxorubicin had a significant decrease in R-wave amplitude, compared with values for the control group, from 30 to 210 mg/m(2). Doxorubicin-treated dogs had decreases in fractional shortening and left ventricular ejection fraction evident as early as 30 mg/m(2), but significant differences between groups were not detected until 90 mg/m(2)was reached. There was also a significant increase in PRA (>or= 120 mg/m(2)) and left ventricular end-systolic and end-diastolic dimensions (>or= 60 and >or= 180 mg/m(2), respectively). Systemic arterial pressure, remaining echocardiographic variables, and concentrations of norepinephrine and BNP had significant variations, but of no clinical importance, during doxorubicin administration. Doxorubicininduced cardiotoxicosis developed at 120 mg/m(2), but there were no clinical signs of dilated cardiomyopathy or congestive heart failure. Echocardiography and determination of PRA were able to detect early cardiac alterations during the development of dilated cardiomyopathy, despite apparently differing degrees of sensitivity to development of doxorubicin-induced cardiotoxicosis.

Matrix-Assisted Laser Desorption/Ionization, Time of Flight, and Angiotensin II

Elsewhere in this issue, Jankowski et al show that mononuclear cells produce sufficient angiotensin II (Ang II) to stimulate Ang II type 1 receptors.1 That finding alone is noteworthy; however, my attention was riveted by the methods the authors used to measure Ang II. Ang II is generally measured by radioimmunoassay. Because of antibody specificity, the samples are first purified by high-performance liquid chromatography (HPLC). The assay is difficult, tedious, and fraught with pitfalls. The sample collections, standardization of collection conditions, and use of appropriate inhibitors are only some of the problems. Only a few investigators do this assay well. An alternative, more accurate, method would be most welcome. Were the method sufficiently established, and were the method routinely available, direct Ang II measurements could be of great clinical utility. After reading the article and meeting with the senior author, I hedge the hope that matrix-assisted laser desorption/ionization (MALDI)–time of flight (TOF) might bring this wish to reality. Jankowski et al adapted MALDI-TOF to measure Ang II or other small peptides and proteins with interesting biological activities in a previous study.2 In their article, they outlined a MALDI-TOF–based strategy to screen protein fractions for defined enzymatic activities. The authors prepared porcine kidney extracts that were then immobilized by covalent coupling to activated affinity beads. The immobilized proteins were incubated with probes …

The interaction of plasma renin activity and plasma atrial natriuretic peptide in 21-hydroxylase deficiency patients

Plasma renin activity (PRA) determination is the main index used to evaluate the mineralocorticoid control in 21-hydroxylase deficiency (21-OHD). PRA values within or at the upper limit of the age-appropriate range, or values <5 or 10 ng/ml/h have been regarded as adequate control. Atrial natriuretic peptide (ANP) has opposite actions to those of angiotensin II/aldosterone, and could help to understand the hydrosaline homeostasis in 21-OHD. We studied the interaction between PRA and ANP levels in 10 controls and 26 patients with 21-OHD under corticoid treatment. Patients were divided into two groups according to PRA levels, < or > or = 5 ng/ml/h, irrespective of the clinical form of 21-OHD. Blood samples for determination of PRA and ANP levels were taken after 30 min in the sitting position (basal), after 30 min in the recumbent position and after 15 min of 20 degrees head-down tilting. ANP levels (pg/ml) in the basal, supine and after head-down tilting position were 25.9 +/- 1.6, 42.7 +/- 7.4 and 54.3 +/- 5.5 in controls; 28.5 +/- 2.1, 38.3 +/- 2.1 and 48.8 +/- 4.1 in the group with PRA levels <5 ng/ml/h, and 20.9 +/- 1.9, 26.6 +/- 2.5 and 34.6 +/- 3.1 in the group with PRA levels > or = 5 ng/ml/h, respectively. Basal and after head-down tilting ANP plasma levels were similar between the controls and the group with PRA levels <5 ng/ml/h. However, the group of patients with PRA levels > or = 5 ng/ml/h showed lower basal and stimulated ANP levels compared to the control group (p<0.05). The decreased plasma ANP levels in the basal condition and after head-down tilting indicate a chronic contraction of the extracellular volume in 21-OHD patients with increased PRA levels. Therefore, mineralocorticoid deficiency is counteracted by decreased ANP secretion in order to preserve fluid and electrolyte homeostasis.

Plasma cathepsin D isoforms and their active metabolites increase after myocardial infarction and contribute to plasma renin activity

Plasma renin activity (PRA) is often found to increase after myocardial infarction (MI). Elevated PRA may contribute to increased myocardial angiotensin II that is responsible for maladaptive remodeling of the myocardium after MI. We hypothesized that MI would also result in cardiac release of cathepsin D, a ubiquitous lysosomal enzyme with high renin sequence homology. Cathepsin D release from damaged myocardial tissue could contribute to angiotensin formation by acting as an enzymatic alternate to renin. We assessed circulating renin and cathepsin D from both control and MI patient plasma (7-20 hours after MI) using shallow gradient focusing that allowed for independent measurement of both enzymes. Cathepsin D was increased significantly in the plasma after MI (P < 0.001). Furthermore, circulating active cathepsin D metabolites were also significantly elevated after MI (P < 0.04), and contained the majority of cathepsin D activity in plasma. Spiking control plasma with cathepsin D resulted in a variable but significant (P = 0.005) increase in PRA using a clinical assay. We conclude that 7-20 hours after MI, plasma cathepsin D is significantly elevated and most of the active enzymatic activity is circulating as plasma metabolites. Circulating cathepsin D can falsely increase clinical PRA determinations, and may also provide an alternative angiotensin formation pathway after MI.

Differential effect of acute angiotensin II type 1 receptor blockade on the vascular and adrenal response to exogenous angiotensin II in humans

Background Aldosterone stimulation by angiotensin II may not exclusively be mediated by the angiotensin II type 1 (AT 1) receptor. We have, therefore, investigated the vascular and adrenal response to angiotensin II infusion without and with pretreatment with the AT 1 receptor antagonist valsartan (160 mg). Methods In nine healthy human volunteers, angiotensin II was administered intravenously at doses of 1, 3, and 10 ng/kg/min, each over 45 min. Arterial blood pressure (BP) was measured oscillometrically at 5-min intervals. Blood for the determination of plasma renin activity and aldosterone was taken before the start of the infusion, at the end of each infusion period, and 1 h after the infusion was stopped. Results Angiotensin II increased systolic and diastolic BP from 121 ± 3/70 ± 2 mm Hg to a maximum of 146 ± 2/97 ± 1 mm Hg ( P < .001) and plasma aldosterone from 39.2 ± 9.8 to 290.7 ± 48.3 ( P < .001). The increase in BP after exogenous angiotensin II was completely abolished in volunteers pretreated with valsartan, averaging 118 ± 3/72 ± 1 mm Hg by the end of the maximum angiotensin infusion dose. In contrast, plasma aldosterone stimulation by angiotensin II was only partially blunted by concomitant AT 1 receptor blockade (98.9 ± 16.3 pg/mL after the maximal dose of angiotensin II). Conclusions These results indicate that although the vascular response to exogenous angiotensin II is exclusively mediated by the AT 1 receptor, the effects of angiotensin II on adrenal aldosterone release may involve other pathways.