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Clinical Chemistry 45: 659-664, 1999;
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(Clinical Chemistry. 1999;45:659-664.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

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

Victoria F. Fredline1, Eva M. Kovacs2, Paul J. Taylor2 and Anthony G. Johnson2,a

1 Department of Clinical Pharmacology, Princess Alexandra Hospital, Brisbane, Queensland, Australia 4102.

2 University of Queensland Department of Medicine, Princess Alexandra Hospital, Brisbane, Queensland, Australia 4102.
a Address correspondence to this author at: James Lance Glaxo Wellcome Medicines Research Unit, Parkes 10 East, Prince of Wales Hospital, Randwick, New South Wales, Australia 2031. Fax 61-2-9382 4053; e-mail agj34419{at}glaxowellcome.co.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: 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).

Methods: After incubation (37 °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 µL/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).

Results: 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.

Conclusion: 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.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Renin is a proteolytic enzyme released from the juxtaglomerular cells of the kidney. Renin acts on its hepatic substrate, angiotensinogen, to produce the decapeptide, angiotensin 1 (Ang1).1 Ang1 is further cleaved by angiotensin-converting enzyme to form the octapeptide, angiotensin 2 (Ang2) (1). Ang2 is one of the most potent vasopressors in humans and plays an important role in blood pressure regulation (2)(3). The direct measurement of Ang2 is difficult because of its very low circulating concentrations (3)(4) and extremely short in vivo half-life as it is rapidly degraded to inactive polypeptide fragments by angiotensinases in plasma and tissue (2). The plasma renin activity (PRA), measured by quantifying the Ang1 generated when plasma is incubated with endogenous renin substrate in the presence of converting enzyme inhibitors, can be used to reflect the activity of the renin-angiotensin system and may be useful in the diagnosis and management of hypertension (1).

Several methodologies for the analysis of PRA have been reported. These include RIA (1)(5)(6)(7)(8)(9)(10), HPLC-RIA (2)(11)(12), and HPLC (13)(14)(15). RIA methods are based on competitive binding principles, and the antibodies used can undergo nonspecific binding with other endogenous angiotensins. This potential cross-reactivity can cause overestimation of the activity and represents a clear disadvantage of RIA. To overcome this potential problem, other investigators have used HPLC to isolate Ang1 from other angiotensins before quantification with RIA, but these methods are extremely labor-intensive because of the additional purification steps involved. Some HPLC methods for the quantification of Ang1 have been developed using ultraviolet (13) and fluorescence detection (14). However, the improved specificity of these methods is negated by their sensitivity, which is inadequate to quantify clinically relevant PRA values.

The relatively recent availability of HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) as an analytical tool has provided specific quantification of compounds at much lower concentrations than conventional HPLC (16). We report here a highly specific HPLC-ESI-MS/MS method with a limit of detection of 0.50 ng Ang1 on the column, which when used in conjunction with the optimized PRA incubation procedure reported by Sealey (1), provides a sensitive and specific measure of PRA at clinically important concentrations (PRA >=0.14 ng Ang1 · mL-1 · h-1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reagents and glassware
Ang1 (Fluka Biochemicals); [Val]5Ang1, phenylmethylsulfonyl fluoride (PMSF), and soybean trypsin inhibitor (Sigma Chemical Co.); and maleic acid and neomycin sulfate (ICN Chemicals) were purchased from their respective suppliers. Doubly distilled deionized water (MilliQ; Millipore Corp.) and HPLC-grade methanol (Merck) were used throughout the investigation. Stock solutions of Ang1 (0.250 mg/mL) and [Val]5Ang1 [0.10 mg/mL; internal standard (IS)] were prepared in methanol. A working solution of IS (2 µg/mL) was prepared in water from the stock solution. Working solutions of maleic acid (276 mmol/L), neomycin sulfate (100 g/L), soybean trypsin inhibitor (2 µg/L), and ammonium acetate buffer (40 mmol/L) were prepared in water. The PMSF solution (1.5 mmol/L) was prepared in methanol. Before use, all glassware was treated with a 50 g/L solution of dichlorodimethylsilane (Sigma) in toluene (Mallinkrodt) to prevent adsorption of the Ang1 onto the glass.

calibrators and controls
From the Ang1 stock solution (0.250 mg/mL) a series of working calibrators (2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 250.0, and 500.0 ng/mL) and blank samples were prepared in normotensive pooled plasma. Aliquots (750 µL) of these calibrators were stored frozen (-20 °C) in 1.5-mL polypropylene microcentrifuge tubes. The calibrators were not incubated; therefore, only the minimal endogenous Ang1 contributed to the calibration curve. This could be accounted for by the subtraction of a blank. Controls were prepared by pooling patient plasma into four groups according to their PRA values obtained using RIA analysis. The four controls contained high, intermediate, low, and very low renin activity. These controls were run within each sample batch throughout the study for quality assurance.

handling of patient samples
All patient samples used in this study were collected into purple-capped EDTA Vacutainer Tubes. The tubes were kept at room temperature and not placed on ice because cooler temperatures favor the cryoactivation of prorenin to renin. The whole blood samples were centrifuged at room temperature (5 min at 2800g), and the plasma was transferred to 5-mL polystyrene tubes and stored frozen at -20 °C until analysis.

hplc-esi-ms/ms conditions
The HPLC system consisted of a Waters 616 solvent delivery system and 600S controller (Waters) and an ISS 200 autoinjector (Perkin-Elmer). Chromatography was performed using a Waters Novapak C18 column (2 x 150 mm, 4 µm) at ambient temperature. The mobile phase consisted of 200 mL/L ammonium acetate buffer (40 mmol/L, pH 5.1)–800 mL/L methanol, delivered at a flow rate of 150 µL/min. The mobile phase was split 1:3 postcolumn into the mass spectrometer. A 20-µL aliquot of the extracted sample was injected for analysis.

A PE-SCIEX API III tandem mass spectrometer (PE-SCIEX), in selected reaction monitoring (SRM) mode, was used for quantitative mass spectrometric detection; the dwell time was 800 ms, and the scan rate was 0.42/s. An electrospray (pneumatically assisted ion spray) interface was operated in positive ionization mode. The orifice potential and interface temperatures were set at 60 V and 60 °C, respectively. Argon, at a density of 300 x 1012 molecules/cm2, was used as the collision gas. The peak-area ratio of Ang1 to IS was used for quantification. Calibration curves were constructed by using weighted (1/x2) linear least-squares regression. Data were collected and manipulated on Macintosh computers operating RAD and MACQUAN software programs (PE-SCIEX). The latter program automatically calculated the signal and noise to allow determination of the limit of detection, defined as a signal-to-noise ratio of 3:1.

incubation procedure
Frozen plasma samples and controls were thawed rapidly in front of a fan for <10 min to prevent cryoactivation of prorenin to renin. Duplicate aliquots (0.5 mL) were pipetted into 1.5-mL polypropylene microcentrifuge tubes. Maleic acid (100 µL), neomycin sulfate (10 µL), and PMSF (5 µL) working solutions were added, and the samples were mixed. One of each duplicate sample was incubated for 3 h (37 °C) and the other for 18 h (37 °C). Samples were frozen (-20 °C) to stop the incubation and stored at this temperature until analysis.

extraction procedure
Frozen calibrators, incubated controls, and incubated patient samples were thawed rapidly in front of a fan for <10 min. Maleic acid working solution (100 µL) and IS (100 µL) were added to each calibrator, control, and patient sample. Samples were mixed and centrifuged (5 min at 20 800g). Solid-phase extraction cartridges (C18, 100 mg, 1 mL; Waters) were preconditioned with methanol (4 mL) followed by water (4 mL). The supernatants were applied to their respective cartridges and drawn through under reduced pressure (20 psi). The loaded cartridges were washed with water (2 mL). Samples were eluted with methanol (2 mL), evaporated to dryness under nitrogen (37 °C), and reconstituted in mobile phase (50 µL).

validation of hplc-esi-ms/ms assay
A calibration curve (2.5, 5, 10, 25, 50, 100, 250, and 500 ng Ang1/mL) was prepared daily with pooled patient sera control samples (PRA = 26.1, 13.5, 3.2 and 0.78 ng Ang1 · mL-1 · h-1). The imprecision (CV) of the assay was determined using the method of Krouwer and Rabinowitz (17) with quadruplicate analysis of each control over 4 days (n = 16). The order in which controls and calibrators were analyzed was randomized to remove positional bias. Absolute recoveries were calculated by comparing the peak areas of the extracted calibrators to solutions of the Ang1 calibrators and IS in mobile phase (n = 4).

assessment of the effect of incubation time on the hplc-esi-ms/ms pra assay
Patient sera (n = 33) were assayed after incubation for 3 and 18 h. The PRA results, calculated from these two different incubation times, were compared to study the effect of incubation time on the HPLC-ESI-MS/MS PRA results. These results were compared using the procedure of Bland and Altman (18). These data were expressed as the mean difference ± SE.

assessment of pra assays: hplc-esi-ms/ms vs ria
Patient sera (n = 31) were assayed using HPLC-ESI-MS/MS and the GammaCoat® plasma renin activity 125I RIA kit (INCSTAR Corp.). For the HPLC-ESI-MS/MS assay, each sample was incubated for both 3 and 18 h and then analyzed. The PRA results for comparison were quantified from the samples incubated for 3 h. RIA samples were incubated for 1.5 h, according to the manufacturer's specifications (19). The results for the patient samples were compared between the two assays (3-h incubation for HPLC-ESI-MS/MS and 1.5-h incubation for RIA), using the procedure of Bland and Altman (18). These data were expressed as mean difference ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
validation of the hplc-esi-ms/ms assay
The IS was selected because of its chemical and structural similarity to Ang1. The IS is a synthetic analog of Ang1 (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) with a valine residue (methyl side group) substituted for the isoleucine residue (ethyl side group) at position five and a mass difference of 14 atomic mass units (CH2+). The acidic mobile phase (pH 5.1) allowed the protonation of the side chains of the two histidine amino acids in both the Ang1 and IS, producing stable [M + 2 H]2+ precursor ions at m/z 649.0 and 641.9, respectively. The characteristic collision-assisted dissociation spectra of Ang1 and IS are shown in Fig. 1 . The predominant precursor and product ions for Ang1 (m/z 649.0->784.0) and IS (m/z 641.9->770.4) were selected and optimized for SRM. Ang1 and IS both fragment at the same position to form the singly charged hexapeptides (1–6)Ang1 and (1–6)IS, respectively. Fig. 2 shows SRM chromatograms of an Ang1 calibrator (2.5 ng/mL, 0.50 ng on column) and a patient plasma sample (PRA = 0.62 ng Ang1 · mL-1 · h-1, 2.23 ng Ang1 on column). The retention times of Ang1 and IS were 2.9 and 2.8 min, respectively, with a total chromatographic analysis time of 5 min per sample.



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  		Figure 1. Collision-assisted dissociation spectra for Ang1 (m/z 649.0->784.0; A) and IS (m/z 641.9->770.4; B).



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  		Figure 2. SRM chromatograms of Ang1 (solid line) and IS (dotted line) for Ang1 calibrator (2.5 ng/mL; A) and a patient plasma sample (PRA = 0.62 ng Ang1 · mL-1 · h-1; B).

The assay was linear over the range 2.5–500 ng/mL, with weighted regression (1/x2) producing a line of best fit of: y = 3.28 x 10-3 (± 3.95 x 10-4)x + 7.62 x 10-3 (± 2.00 x 10-3), and a correlation coefficient of 0.994 (n = 4). The limit of detection (signal-to-noise ratio of 3:1) of Ang1 was 0.50 ng on the column (PRA = 0.14 ng Ang1 · mL-1 · h-1). The imprecision of the assay was determined at four activities (PRA = 26.1, 13.5, 3.2, and 0.78 ng Ang1 · mL-1 · h-1) with total CVs of 7.0%, 7.0%, 15%, and 11%, respectively (Table 1 ). Absolute recoveries were 92.3% (67.9–112.9%) and 87.4% (63.6–104.9%) for Ang1 and IS, respectively (n = 4).


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  		Table 1. Imprecision of the HPLC-ESI-MS/MS assay (n = 16).

comparison of incubation times (3 and 18 h)
A total of 33 patient samples were incubated for both 3 and 18 h and assayed using HPLC-ESI-MS/MS. The data showed linear correlation (r = 0.982; 18 h = 0.872 x 3 h + 0.147; Fig. 3 A). Fig. 3B shows the data as the differences between PRA measured after 3- and 18-h incubations vs the mean. The mean difference (mean bias) of these PRA results was 0.0172 ng Ang1 · mL-1 · h-1 (Sy|x = 0.0406 ng Ang1 · mL-1 · h-1) over the activity range 0.22–5.08 ng Ang1 · mL-1 · h-1.



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  		Figure 3. PRA results obtained using HPLC-ESI-MS/MS after 3-h vs 18-h incubation (A) and differences between plasma samples incubated for 3 and 18 h vs the mean result (B).

(A), n = 33; r = 0.982. The line of identity (solid line) and the line of best fit [dotted line; PRA (18 h) = 0.872 x PRA (3 h) + 0.147] are shown. (B), the mean difference (solid line), mean ± 1 SD (dashed lines), and mean ± 2 SD (dotted lines) are shown.

comparison of plasma pra: hplc-esi-ms/ms vs ria
A total of 31 patient samples were assayed using HPLC-ESI-MS/MS (3-h incubation) and RIA (1.5-h incubation). These data showed a linear correlation (r = 0.959; RIA = 2.16 x HPLC-ESI-MS/MS - 0.247; Fig. 4 A). Fig. 4B shows the data as the differences between PRA measured using RIA and HPLC-ESI-MS/MS vs the mean of both methods. The mean difference (mean bias) of these PRA results was 1.07 ng Ang1 · mL-1 · h-1 (Sy|x = 0.254 ng Ang1 · mL-1 · h-1) over the activity range 0.22–10.5 ng Ang1 · mL-1 · h-1.



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  		Figure 4. PRA results obtained using HPLC-ESI-MS/MS vs RIA (A) and differences between RIA and HPLC-ESI-MS/MS PRA results vs the mean result (B).

(A), n = 31; r = 0.959. The line of identity (solid line) and the line of best fit (dotted line; RIA = 2.16 x HPLC-ESI-MS/MS - 0.247) are shown. (B), the mean difference (solid line), mean ± 1 SD (dashed lines), and mean ± 2 SD (dotted lines) are shown.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As discussed previously, the current methods of determining PRA have involved RIA, HPLC, or a combination of both techniques. RIA and HPLC procedures have limitations in specificity and sensitivity, respectively, whereas RIA-HPLC methods are too time-consuming for routine analysis because of the additional purification steps required. In using HPLC-ESI-MS/MS, we have developed a sensitive and specific measurement of PRA through the quantitative analysis of Ang1 in plasma.

An alternative to the measurement of PRA is the determination of the active renin concentration (ARC). In this case, the concentration of active renin, the renin capable of converting angiotensinogen to Ang1, is quantified directly in plasma by the use of monoclonal antibodies (20). In contrast, PRA quantifies the product of renin action. Some authors (6)(7)(8)(9)(20) advocate the measure of ARC over PRA because of the longer analysis times required for PRA (6) and the poor interlaboratory reproducibility of PRA reported for the multicenter comparative study (20). However, the PRA assay has several advantages over ARC. First, PRA is the more important clinical indicator because the normal circulating concentration of angiotensinogen is close to the KM of the reaction, which allows renin to generate Ang1 at only one-half the maximal velocity. Therefore, the concentration of angiotensinogen will affect renin activity (1). For this reason, PRA represents the capacity of plasma renin and angiotensinogen to generate the active hormone Ang2 (via Ang1) and does not necessarily correlate with ARC. This is highlighted by the fact that comparative studies of PRA and ARC at clinically relevant concentrations gave poor correlations [r = 0.599; PRA <2.5 ng Ang1 · mL-1 · h-1 (7); and r = 0.687; PRA <5 ng Ang1 · mL-1 · h-1 (8)] and demonstrates that ARC is not a suitable indicator of Ang2 production. Second, PRA has the advantage that higher sensitivity can be achieved by prolonging the incubation time, which allows each renin molecule to cleave many Ang1 molecules from angiotensinogen.

Although the Italian multicenter comparative study reported poor reproducibility of PRA (20), Sealey (1) has reported optimal sample handling and incubation conditions to minimize variability of the assay. These include the prevention of cryoactivation of prorenin; optimization and control of pH and incubation times; and the addition of PMSF, EDTA, and neomycin solution to inhibit angiotensinases, angiotensin-converting enzyme, and microbial growth (1). If these conditions are controlled, angiotensinases can be successfully inhibited for up to 18 h, and these extended incubation times negate the necessity of blank subtraction, a major source of variability in some PRA assays (1).

The limited linear range of the existing RIA methods means that samples with high renin (13 < PRA < 44 ng Ang1 · mL-1 · h-1) must be diluted fivefold and the assay repeated (4). Sealey and co-workers (4)(5) have reported that the rate limiting of PRA by angiotensinogen concentration is nonlinear. Plasmas from different individuals have different concentrations of angiotensinogen (renin substrate). Because the enzymatic conversion of angiotensinogen to Ang1 obeys first-order kinetics, a dilution of one patient plasma may halve the amount of Ang1 generated, whereas a similar dilution of a sample in which the substrate concentration is greater may not cause a proportional reduction in the amount of Ang1 generated (5). Thus, sample dilution lowers the concentration of renin substrate and alters the PRA values such that they cannot be calculated by multiplication with a dilution factor. It is therefore recommended that dilution of samples should be avoided if possible (5). The HPLC-ESI-MS/MS assay is linear up to 500 ng/mL Ang1 (PRA = 167 ng Ang1 · mL-1 · h-1), which negates the need for sample dilution of high renin samples.

There has been some dispute as to whether PRA remains linear at incubation times up to 18 h (21). Sealey et al. (4) have recommended both 3 and 18 h incubation of all samples, providing that incubation conditions are controlled carefully. Samples with PRA >0.64 ng Ang1 · mL-1 · h-1 are reported after 3-h incubations, whereas samples with PRA <0.64 ng Ang1 · mL-1 · h-1 are quantified after 18-h incubations. This avoids substrate exhaustion, which can potentially cause a drop off in renin activity at higher PRA, but still provides greater sensitivity for lower PRA samples. Sealey et al. (5) compared PRA results from samples (n = 32; PRA<1.8 ng Ang1 · mL-1 · h-1) analyzed after 3- and 18-h incubations, and the data were linearly correlated [r = 0.985; PRA (18 h) = 0.90 x PRA (3 h) + 0.07]. In this study, we found a similar correlation for PRA results over the same range [n = 26; PRA <2.0 ng Ang1 · mL-1 · h-1; r = 0.962; PRA (18 h) = 0.960 x PRA (3 h) + 0.105]. However, when the PRA range is extended up to PRA <5 ng Ang1 · mL-1 · h-1, the regression line deviates from the line of identity, indicating lower PRA results after an 18-h incubation. This may be because of substrate exhaustion in samples with higher PRA and indicates why samples with PRA <0.64 ng Ang1 · mL-1 · h-1 should be analyzed after incubation for 3 h.

Comparison of the PRA results obtained with the HPLC-ESI-MS/MS method vs RIA (Fig. 4AUp ) shows that the PRA results obtained with RIA were on average higher than those obtained with HPLC-ESI-MS/MS. Although different calibrators were used for each assay, this may account for some, but not all, of the discrepancies observed. This highlights the potential overestimation of RIA, which may be attributed to several possible factors. These include cross-reactivity of the antibodies with other endogenous compounds in the plasma, deterioration of the kit calibrators through loss of activity from storage in dilute solution (5), errors introduced through blank subtraction, or dilution of the plasma samples, which dilutes the substrate, which in turn has a nonlinear effect on the generation of Ang1 (5). A plot of the differences between the RIA and HPLC-ESI-MS/MS PRA results vs the mean PRA (Fig. 4BUp ) shows a systematic increase in the results from the RIA relative to the results from HPLC-ESI-MS/MS with increasing PRA. The clinical importance of this overestimation of PRA by RIA is substantial given that a doubling of PRA would halve the aldosterone:renin ratio and may lead to misdiagnosis of patients with primary hyperaldosteronism. In addition, overestimation of PRA by RIA may imply that patients with hypertension have high renin, and could influence the choice of antihypertensive medication prescribed for a patient.

In conclusion, we have developed a sensitive and specific HPLC-ESI-MS/MS method for the analysis of PRA. Comparison of this method with RIA showed that, on average, RIA PRA results were higher than those obtained with HPLC-ESI-MS/MS. This difference could be clinically important in the diagnosis of hypertensive patients.


   Acknowledgments
 
We thank Ross Rappel and Greg Ward from the Endocrine Section, Chemical Pathology, Princess Alexandra Hospital, for the RIA PRA measurements for the comparative study. We also thank Lambro Johnson, Chemical Pathology, Royal Brisbane Hospital, Queensland, Australia, for providing information on the INCSTAR 125I RIA kit.


   Footnotes
 
1 Nonstandard abbreviations: Ang1, angiotensin 1; Ang2, angiotensin 2; PRA, plasma renin activity; ESI-MS/MS, electrospray-tandem mass spectrometry; PMSF, phenylmethylsulfonyl fluoride; IS, internal standard; SRM, selected reaction monitoring; and ARC, active renin concentration.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. Rauh, M. Groschl, and W. Rascher
Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography-Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants
Clin. Chem., May 1, 2007; 53(5): 902 - 910.
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