Plasma Renin Activity Assay Research Articles

Correlation between plasma renin concentration and plasma renin activity assays on the Maglumi 800 chemiluminescence immunoassay analyzer

The ratio of plasma aldosterone concentration to plasma renin activity (PAC/PRA) is the most common screening test for primary hyperaldosteronism, but it is not standardized among laboratories. We evaluated the correlation between plasma renin concentration (PRC) and plasma renin activity (PRA), and then PAC/PRC and PAC/PRA ratios on Maglumi 800 immunoassay analyzer. Forty-five plasma samples from volunteers were collected. PRC, PRA and PAC were measured by Maglumi 800 - a fully-auto chemiluminescence immunoassay analyzer. Correlation between plasma renin concentration (PRC) and plasma renin activity (PRA), and then PAC/PRC and PAC/PRA ratios were determined by regression analysis. PRC and PRA showed a good correlation (r = 0.88). The PAC/PRC and PAC/PRA ratios have a similar relationship (r = 0.757). Because of the advantages of the procedure and the independence of PRC from endogenous angiotensinogen levels, PRC may be used preferentially over PRA as high throughput screening tool.

Commutability assessment of candidate reference materials for plasma renin activity measurement: current challenges.

This study aims to evaluate the commutability of external quality assessment (EQA) materials and candidate reference materials (RMs) for plasma renin activity (PRA) assay. Commutabilities of 16 candidate RMs were measured along with 40 clinical samples by the four different routine PRA assays, including three LC‒MS/MS assays and onechemiluminescence immunoassay. Sixteen candidate RMs included native/spiked human plasma pools (small-scale pools with <50 individuals) and current EQA materials (large-scale pools with >1,000 individuals). Difference inbias approach and linear regression with prediction interval approach were adopted to determine the commutability. Two-way variance analysis was used to estimate the effects of spiked and pool size on the commutability. Stability and homogeneity studies were performed. Precision and correlation performance of all assays was acceptable. In the difference in bias approach, thecommutability results were not satisfactory (noncommutability: 14/16) and significant sample-specific effects were detected in assay pairs using different incubation buffers. For the prediction interval approach, no commutability was observed in the spiked small-scale pools; EQA materials (4/9) had more satisfactory commutability among all assays than the small-scale pools (2/7); RMs of large-scale pools tend to have better commutability no matter spiked ornot. Commutable RMs were obtainable but challenging. Current EQA materials with relatively good commutability, stability, and homogeneity were appropriate RMs. Large-scale pools are tending to be commutable. Spiking in small-scale pools was not suggested to prepare RMs. MPs adopting a uniform incubation buffer would be preferable for further commutability research.

ODP596 Central Adrenal Insufficiency After Intra-Articular Triamcinolone Injection In A Patient On Dolutegravir And Cobicistat Boosted Darunavir

Abstract Background Central adrenal insufficiency from drug-drug interaction between steroids and HIV anti-retroviral medications is a well-documented phenomenon in the literature, but in clinical practice making this diagnosis can prove challenging. Clinical Case 58-year-old woman with well-controlled HIV on dolutegravir and cobicistat/darunavir, type 2 diabetes mellitus, and chronic kidney disease presented to our emergency department with 3 days of polyuria, polydipsia, headaches, nausea, and decreased appetite. She also noticed that her glucose readings have been much higher than usual. 5 days prior to presentation, she received a 40 mg triamcinolone acetonide injection into her right subacromial bursa for chronic shoulder pain. For diabetes, she had been on the same dosage of weekly dulaglutide with good control. Initial laboratory evaluation revealed elevated serum glucose of 763 mg/dl (70 - 140 mg/dL) without an elevated anion gap, sodium of 124 mEq/L (135 - 145 mEq/L) and potassium of 5.9 mmol/L (3.6 - 5.2 mmol/L). Morning cortisol was sent as part of the workup for hyponatremia and returned low at 1.2 ug/dL (4 - 20 ug/dL). A 250 mcg cosyntropin stimulation test was performed, with baseline cortisol at 1.3 ug/dL and one-hour post-stimulation cortisol of 8.8 ug/dL. Pre-stimulation ACTH was &amp;lt; 5. 0 pg/mL (10 - 60 pg/mL). A synthetic glucocorticoid panel were sent on the 11th day after her injection and detected triamcinolone acetonide at 3.3 mcg/dL (cutoff: 0.1 mcg/dL) in urine and 0.85 mcg/dL (cutoff: 0.1 mcg/dL) in serum. She was diagnosed with iatrogenic adrenal insufficiency from intra-articular triamcinolone injection, the effect of which had been potentiated by her HIV medications cobicistat and dolutegravir (both CYP3A4 inhibitors). She was started on hydrocortisone and experienced improvement in appetite and fatigue. A plasma renin activity assay was elevated at 7.66 ng/mL/h (0.25 - 5.82 ng/ml/h) with 15 ng/dL of aldosterone (3 - 16 ng/dL). While awaiting the ACTH result, we discussed the possibility of primary adrenal insufficiency given persistent hyponatremia despite improved glucose control. However, given recent normal cross-sectional abdominal imaging, the history of steroid exposure, and lack of opportunistic infections in the past, central adrenal insufficiency remained the leading diagnosis. Further workup, including urine anion gap, eventually determined the cause of her hyponatremia to be a type 4 RTA likely related to her longstanding history of diabetes. The patient was discharged home on hydrocortisone with plan for repeat stimulation test in 6 weeks. Conclusion 1. Keep primary adrenal insufficiency in mind in a patient with HIV who presents with hyponatremia and low cortisol. 2. Steroid injections carry risks of significant hyperglycemia and iatrogenic adrenal insufficiency in patients receiving cobicistat and integrase inhibitor. Presentation: No date and time listed

Overt and Subclinical Adrenal Insufficiency in Pulmonary Tuberculosis

BackgroundTuberculosis of the adrenal glands may cause overt or subclinical adrenal insufficiency. An algorithm-based approach including assessment of paired basal cortisol and plasma adrenocorticotropic hormone (ACTH), short Synacthen, and plasma renin activity assays could be useful to diagnose all forms of adrenal insufficiency. MethodsThis cross-sectional study included consecutive, treatment-naive subjects diagnosed with pulmonary tuberculosis. Tuberculosis severity was classified by radiological criteria. Baseline parameters plus morning (8 am) serum cortisol and paired plasma ACTH were measured in all patients. Synacthen stimulation tests and plasma renin activity assays were performed as required. ResultsEighty-four treatment-naive consecutive cases of pulmonary tuberculosis were evaluated for adrenal insufficiency. Twenty-seven (32.14%) subjects had normal adrenocortical function and 8 (9.5%), 7 (8.3%), 40 (47.6%), and 2 (2.4%) subjects had stage 1, stage 2, stage 3, and stage 4 adrenal insufficiency, respectively. Serum cortisol was negatively correlated with radiological severity (P = .01) and duration of illness (P = .001). Adrenal dysfunction was present in 27.3%, 82.5%, and 80% of those with radiologically minimal, moderately advanced, and far-advanced disease, respectively. Mean cortisol was 19.74 ± 5.52, 17.42 ± 8.53, and 15.71 ± 7.14 (μg/dL) in the 3 groups, respectively (P = .042). Hyponatremia was present in 83.3% of the patients. Serum sodium was negatively correlated with severity but not with the duration of disease. ConclusionThe prevalence of overt and subclinical adrenal dysfunction in pulmonary tuberculosis was high and was correlated with disease severity and duration. An algorithmic approach may be useful to detect the same and may have important clinical implications.

Measurement of Equilibrium Angiotensin II in the Diagnosis of Primary Aldosteronism.

Many medications (including most antihypertensives) and physiological factors affect the aldosterone/renin ratio (ARR) when screening for primary aldosteronism (PA). We sought to validate a novel equilibrium angiotensin II (eqAngII) assay and compare correlations between the aldosterone/angiotensin II ratio (AA2R) and the current ARR under conditions affecting the renin-angiotensin system. Among 78 patients recruited, PA was excluded in 22 and confirmed in 56 by fludrocortisone suppression testing (FST). Peripheral levels of eqAngII, plasma renin activity (PRA) and direct renin concentration (DRC) were measured. EqAngII showed good consistency with DRC and PRA independent of PA diagnosis, posture, and fludrocortisone administration. EqAngII showed close (P < 0.01) correlations with DRC (r = 0.691) and PRA (r = 0.754) during FST. DRC and PRA were below their assays' functional sensitivity in 43.9% and 15.1%, respectively, of the total 312 samples compared with only 7.4% for eqAngII (P < 0.01). Bland-Altman analysis revealed an overestimation of PRA and DRC compared with eqAngII in a subset of samples with low renin levels. The AA2R showed not only consistent changes with the ARR but also close (P < 0.01) correlations with the ARR, whether renin was measured by DRC (r = 0.878) or PRA (r = 0.880). Dynamic changes of eqAngII and the AA2R show good consistency and close correlations with renin and the ARR. The eqAngII assay shows better sensitivity than DRC and PRA assays, especially at low concentrations. Whether the AA2R can reduce the impact of some factors that influence the diagnostic power of the ARR warrants further study.

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.

Customisation and validation of a low-volume plasma renin activity immunoassay: Enabling of regulatory compliant determination in paediatric trials

ObjectivesInvestigations of plasma renin activity (PRA) in children are urgently required. Small-volume, regulatory guideline compliant, bioanalytical assays tailored for paediatric application could facilitate to overcome this hurdle. Ethical constraints given e.g. by the European Medicines Agency need to be addressed and reliable data generation in line with Good Clinical Laboratory Practice must be ensured. MethodsA PRA enzyme-linked immunosorbent assay (ELISA) was tailored for paediatric application and validated in the context of the U.S. Food and Drug Administration bioanalytical guideline. Performance verification of the assay was conducted by participation in an interlaboratory ring test, evaluation of incurred sample reanalysis and an application-orientated approach in children. ResultsA five-fold reduction of required plasma volume to 100 μL was achieved without limiting the calibration range. Between-run accuracy and precision varied no more than 5.0% and 6.3%, respectively. No substantial matrix effect was detected and the inter-run precision for parallelism was 11.1%. Stability experiments approved the freeze-thaw stability, short-term stability as well as 37 weeks of long-term stability. The assay successfully participated in the interlaboratory ring test, showing non-inferiority regarding radioimmunoassay (RIA). Moreover, PRA in plasma samples of neonates was successfully determined. Conducted incurred sample reanalysis confirmed the comparability and reliability of the assay with regard to international regulatory bioanalytical guidelines. ConclusionA fit-for-purpose PRA ELISA characterised by low-volume application was successfully established, indicating non-inferiority regarding commonly applied RIAs. Reliability of the regulatory-compliant PRA assay was proven by participation in an interlaboratory ring test and its application in a paediatric population.

Bead-Extractor Assisted Ready-to-Use Reagent System (BEARS) for Immunoprecipitation Coupled to MALDI-MS

Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples. The combination of immunoprecipitation (IP) with MALDI technology delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can be performed either manually (requiring significant time and labor) or automatically (requiring the purchase of a complex liquid-handling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for plasma renin activity (PRA) and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98. The BEARS technology provides convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.

A widely applicable plasma renin activity assay by LC-MS/MS with offline solid phase extraction

The measurement of plasma renin activity using LC-MS/MS is an attractive alternative to radioimmunoassay techniques. A published method for renin activity by LC-MS/MS uses equipment that is not available in all LC-MS/MS laboratories. Here, we present an offline modification to allow users of LC-MS/MS to perform this analysis without specialist equipment. Samples were prepared in duplicate and incubated for 6.5 h and 24 h. Solid phase extraction was performed offline using Waters Oasis MAX ion-exchange 96-well plate. A method comparison was performed between this assay and a previously published assay using on-line solid phase extraction. The offline method for plasma rennin activity gave similar results to the on-line method across the concentration range. Analytical performance was also found to be comparable and the use of a 24 h incubation for low samples was also deemed unnecessary. The method described is an acceptable alternative to a previously published assay which does not require the use of highly specialist equipment and will be applicable to the majority of LC-MS/MS users.

Development And Validation of An Ultra-Sensitive Method For The Measurement of Plasma Renin Activity in Human Plasma Via Lc–MS/MS

Renin catalyzes the conversion of angiotensinogen to angiotensin I (Ang I), the first and rate-limiting step in the renin-angiotensin-aldosterone system. Plasma renin activity (PRA) is an important target engagement biomarker in the clinical development of renin inhibitors. We have developed and validated an improved PRA assay that incorporates an Ang I trapping antibody followed by extraction and quantification of Ang I using a highly sensitive and specific LC-MS/MS method. The following assay performance characteristics were assessed as part of analytical validation: precision, LOQ, spike recovery, dilution linearity, stability, absolute recovery and biological variability. The assay demonstrated excellent performance characteristics. Notably, the sensitivity of the assay was increased 140-fold when compared with a previous enzyme immunoassay-based assay. The improved sensitivity allowed the measurement of >95% PRA inhibition from baseline levels. In addition, we compared the LC-MS/MS-based assay to an enzyme immunoassay-based assay.

Two-site automated chemiluminescent assay for measurement of immunoreactive renin

Measurement of renin is important for the clinical assessment of hypertensive patients and for the screening for primary aldosteronism. The aim of this study was to evaluate the performances of an automated immunoassay for measurement of immunoreactive renin. Functional sensitivity, in vitro stability, and reference values were determined. Method comparison with the plasma renin activity assay was also performed. Our results demonstrate that the Liaison® direct renin assay may assist the clinician in the assessment of hypertensive patients and in the screening for primary aldosteronism.

A screening procedure for primary aldosteronism based on the Diasorin Liaison® automated chemiluminescent immunoassay for direct renin

Primary aldosteronism (PA), the most common secondary cause of hypertension, can be screened for using the aldosterone/renin ratio. This ratio is raised in PA and its accuracy depends on the ability to measure plasma renin at extremely low concentrations. We compared two different procedures for assessing plasma renin. The conventional method, which measures plasma renin activity (PRA), is technically demanding and laborious, and the Diasorin Liaison method, which measures plasma renin concentration (PRC), is an automated immunoassay. Results from each method were used to calculate the aldosterone/renin ratio (ARR) and the performance of the Diasorin Liaison method compared with that of the conventional assay using receiver operator characteristic curves. The analytical and functional sensitivity of the PRC method were 2.1 and 5 microIU/mL, respectively. Intra- and inter-assay precision were <7.2% and 10.4%, respectively. There was significant (9%) prorenin interference. Samples with PRA > 1.0 ng/mL/h showed significant correlation with PRC (r = 0.93; P < 0.05; n = 146); however, with PRA < 1.0 ng/mL/h, no significant correlation occurred (r = 0.14; P < 0.05; n = 79). An aldosterone (pmol/L)/PRC(microIU/mL) ratio of >35, in patients with aldosterone >300 pmol/L, resulted in 100% sensitivity and 93% specificity, when compared with the commonly accepted aldosterone (pmol/L)/PRA (ng/mL/h) ratio of >750, in identifying patients who may suffer from PA. This study indicates the feasibility of using the automated PRC assay as a replacement for the conventional manual PRA assay in calculating the ARR as a first-line screen for PA.

Plasma Renin Activity: An Assay with Ongoing Clinical Relevance

Featured Article: Fyhrquist F, Soveri P, Puutula L, Stenman U-H. Radioimmunoassay of plasma renin activity. Clin Chem 1976;22:250–6.2 In the early 1970s there was a growing need for reliable and clinically useful methods for the determination of the activity of the renin-angiotensin system. Bioassay methods for renin activity, although valuable in early research, were too cumbersome and unreliable for clinical use. RIAs for plasma angiotensin II were of limited clinical value because of their lack of specificity, the need for extraction procedures, and insufficient sensitivity. We therefore developed a sensitive and specific assay of plasma renin activity (PRA)1 . Plasma was acidified with a mixture of hydrochloric acid and the enzyme inhibitor hydroxyquinoline, to ensure optimal incubation pH (6.0) (1) and sufficient inhibition of proteolytic breakdown …

H 034 SPIRONOLACTONE LOWERS BLOOD PRESSURE IN RESISTANT HYPERTENSIVE PATIENTS WITH LOW PLASMA RENIN ACTIVITY

The purpose of this study was to examine the systolic blood pressure and plasma renin activity (PRA) responses to chronic stress in normotensive rats and in rats with one hypertensive parent. Twenty-four male Wistar-Kyoto (WKR) and 24 male F1 offspring of spontaneously hypertensive and WKY rats (BHR) were randomly assigned to 3 groups of 8 each. Experimental (E) animals were subjected to 2 hr daily of shock-shock conflict. Each response produced a 0.2 sec, 0.2–0.4 mA cutaneous electric shock. Failure to respond in 10 sec resulted in a train of 5 shocks (0.2 sec each sec). Yoked animals (Y) received the same shocks as E but had no control over their presentation. Finally, a control group (C) for maturation received no shocks. The E and Y animals were subjected to 14 weeks of conflict and were then monitored an additional 14 weeks in the absence of shock. All animals had their tail cuff blood pressures taken weekly except for 3 times when bloods were obtained for PRA assays. Analysis of blood pressure data revealed that: (1) BHR animals showed more of a blood pressure response to shock than WKY animals; (2) Y animals showed more of a response to conflict than E, especially for the BHR group; and (3) BHR shocked animals remained permanently elevated compared to BHR control animals even in the 14 week post-conflict period during which no shocks were given. Although PRAs for BHR animals were significantly higher than for WKY at the beginning of study, the stress-induced hypertension was associated with either normal or suppressed PRA values, suggesting that the hypertension in these animals is not a high renin hypertension.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

Analytical and clinical performance of an automated chemiluminescent immunoassay for direct renin measurement: comparison with PRA and aldosterone assays

The analytical performance of a fully automated chemiluminescentimmunoassay (CLIA) by Nichols Institute Diagnostic for direct renin, was evaluated. Within-assay imprecision (CV) resulted 5.6% at 4.7 μU/ml; 1.9% at 33.7 μU/ml and 2.3% at 194.9 μU/ml; between-assay imprecision 12.4% at 5.3 μU/ml; 8.7% at 32.4 μU/ml, 8.2%at 90.2 μU/ml, 5.5% at 196.5 μU/ml. Direct renin CLIA showed good linear relationship with direct renin IRMA and with plasma renin activity (PRA). The clinical performance of direct renin CLIA, PRA and aldosterone assays was tested by ROC analysis in patients with heart failure (HF). For mild HF, areas under the curve were: PRA=0.733, direct renin=0.671, aldosterone=0.789; for severe HF: PRA=0.855, direct renin=0.792, aldosterone=0.801. In conclusion, direct renin CLIA measurement showed good analytical performance (shorter turn around time, better precision and practicability than IRMA and PRA) and good clinical performance in HF patients with different severity of disease similar to that of aldosterone and PRA measurement.

Supraregional Interlaboratory Quality-Control Survey for an Immunoradiometric Renin Assay

For the last 20 years, the most widely used method for assessment of the renin-angiotensin system has been the plasma renin activity assay (1). Although the assay gives useful information on the enzymatic function of the renin molecule, the intrinsic characteristics of this method limit its analytical accuracy (2). Immunoradiometric assays for renin, developed in 1985, overcame this limitation of the enzymatic assay because the well-defined monoclonal antibodies provided the means for direct quantification of specific active forms of the enzyme molecule (3)(4). Nevertheless, a critical debate has developed over the use of these assays (5)(6)(7)(8)(9)(10). A pilot study (11)(12) in a limited number of laboratories (eight Italian centers) found satisfactory indexes of precision for a direct immunoradiometric renin assay (Eria Diagnostics, Sanofi-Pasteur). In our previous experience, the inter- and intralaboratory reproducibility indices of this commercial immunoradiometric assay appeared to be better than those that had been achieved with an enzymatic assay (REN-CTK; Sorin Biomedica), with the differences probably being attributable to the greater complexity of the procedure for the latter assay (11). Various reports have been published during the past 30 years describing useful tools to monitor the performance of immunoassays …

Measurement of Plasma Renin Activity with Use of HPLC-Electrospray-Tandem Mass Spectrometry

The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA). After incubation (37 degrees C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer-800 mL/L methanol as the mobile phase. The flow rate was 150 microL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0-->784.0; IS m/z 641.9-->770.4). The assay was linear over the range 2.5-500 ng Ang1/mL, which corresponded to a limit of detection (signal-to-noise ratio of 3:1) of PRA of 0.14 ng Ang1. mL-1. h-1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1. mL-1. h-1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA <2 ng Ang1. mL-1. h-1, but samples with a PRA of 2-5 ng Ang1. mL-1. h-1 gave lower PRA results after incubation for 18 h than after 3 h. We compared the HPLC-ESI-MS/MS assay and an RIA for the determination of PRA, with PRA incubation times of 3 h and 1.5 h, respectively. The mean PRA based on RIA of Ang1 was higher than that obtained using HPLC-ESI-MS/MS. The HPLC-ESI-MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.

Age-related effects on the zona glomerulosa cells of the rat adrenal cortex

Zona glomerulosa cells of the rat adrenal cortex were studied at 2, 6, 12, 18 and 24 months. Adrenals from unstimulated rats were processed for light and electron microscopy and trunk blood was obtained for the assay of plasma renin activity and aldosterone. Similar assays were made in rats stimulated with adrenocorticotropic hormone (ACTH), 30 min before sacrifice. At light microscopy, no major structural changes were observed; the ultrastructure of most organelles was well preserved and the presence of lipofuscin granules in older animals was remarkable. Acid phosphatase activity was observed in lysosomes and some lipofuscin granules. The quantitative study showed an age-related decrease in cell and nuclear volume (p < 0.01 and p < 0.001 respectively) and an increase in volume density of lipid droplets, lipofuscin granules (p < 0.001), lysosomes and Golgi complex; a decrease in volume density of mitochondria and smooth endoplasmic reticulum (p = 0.052) was also observed. Plasma renin activity and aldosterone level were both reduced (p < 0.001). In stimulated animals, an enhanced aldosterone level was obtained which was higher at 12 months and decreased thereafter. These results show an age-related decrease of aldosterone secretion, consistent with structural and ultrastructural data and related to plasma renin decrement. They also show a decline in the response to aldosterone stimulation by ACTH, indicating a lesser functional ability of zona glomerulosa cells in old animals.

More on Renin

In an editorial comment (1) on our paper (2) , Sealey and Laragh erroneously conclude that the IRMA is a step backward from the traditional enzyme-kinetic plasma renin activity (PRA) assay, and that the new IRMA is not suitable for measuring renin because it comeasures 1–2% of prorenin. They regard the traditional PRA assay as an accurate measure of the in vivo production of angiotensin (Ang) II, the physiologically relevant end product of the renin–angiotensin cascade. In fact, the PRA assay measures the concentration of Ang I that is generated in plasma in vitro after a long incubation period (up to 18 h) under artificial conditions. The PRA assay is an indirect assay of renin and is complicated by the fact that Ang I is usually converted to Ang II and degraded into smaller inactive peptides. Ang I-to-II conversion and Ang I degradation are prevented by lowering the pH of plasma and by adding peptidase inhibitors before the incubation step. Whether this is 100% successful, particularly with such long incubation times, has not been formally tested. These difficulties are not encountered in the direct assay of renin by IRMA. The prorenin concentration in plasma is higher than the renin concentration, and comeasurement of even a small percentage of prorenin may therefore lead to a sizable overestimation of renin. The important question is: What is the true magnitude of this problem? Our study was designed to address precisely this question, and the answer is simple: The problem is not important enough to render the IRMA unsuitable for clinical use. In contrast to what is stated in the editorial comment, our study demonstrates that the the new assay can readily distinguish low-, normal-, and high-renin hypertension. There was good agreement with the enzyme-kinetic assay not only in the normal- and high-renin …