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Articles |
Department of Internal Medicine I, University Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
a Author for correspondence. Fax 31 10 4634531; e-mail deinum{at}inw1.azr.nl
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Abstract |
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Methods: Because the inactive form of prorenin converts slowly into an active form at low temperature, we raised the assay temperature from 22 °C to 37 °C, simultaneously shortening the incubation time from 24 to 6 h. The former IRMA was performed in <1 working day with these modifications.
Results: The comeasurement of prorenin as renin was eliminated. Reagents were stable at 37 °C, and the new and old IRMAs were comparable in terms of precision and accuracy. The functional lower limit of the assay (4 mU/L) was below the lower reference limit (9 mU/L). The modified IRMA agreed closely with the activities measured with an enzyme-kinetic assay. Results were not influenced by the plasma concentration of angiotensinogen. At normal angiotensinogen concentrations, the IRMA closely correlated with the classical enzyme-kinetic assay of plasma renin activity.
Conclusion: The modified IRMA, performed at 37 °C, avoids interference by prorenin while retaining the desirable analytical characteristics of the older IRMA and requiring less time.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Enzyme-kinetic measurements of the so-called plasma renin activity (PRA) are often used to assess the in vivo activity of the renin-angiotensin system, rather than the difficult and labor-intensive measurement of Ang II. In the PRA assay, plasma is incubated at 37 °C. During this incubation, renin acts on an endogenous renin substrate, angiotensinogen, to generate Ang I. The degradation of Ang I and its conversion to Ang II are blocked by protease inhibitors that have been added to the plasma. The Ang I generated in vitro is quantified by radioimmunoassay. Because angiotensinogen circulates at a concentration roughly equal to the Km, the rate of Ang I generation in the PRA assay depends as much on the concentration of renin as on that of angiotensinogen. Plasma angiotensinogen concentrations may vary; therefore, PRA is not always a good measure of renin release by the kidney. To overcome this problem, exogenous angiotensinogen can be added to the incubation mixture at a saturating concentration. This is the principle of the enzyme-kinetic plasma renin concentration (PRC) assay.
Recently, our group described the clinical validation of an IRMA for plasma renin (7). This assay measures renin directly, is better to standardize, and is less labor-intensive than the classical enzyme-kinetic PRA and PRC assays. Our report, however, sparked a polemic on the specificity of the new assay (8)(9)(10) because in the IRMA some prorenin is measured as renin, and therefore, the reliability of the assay at low renin concentrations might be insufficient. We therefore set out to improve our method to minimize the comeasurement of prorenin.
We hypothesized that the problem was caused less by cross-reactivity of the renin-specific antibody with native, enzymatically inactive prorenin than by inadvertent activation of prorenin during the assay procedure. It is known that prorenin spontaneously undergoes a conformational change by which it becomes enzymatically active (active prorenin). This change does not require the proteolytic cleavage of the prosegment and does not necessarily lead to generation of renin. The nonproteolytic activation of prorenin is time- and temperature-dependent. Activation occurs mainly at low temperature (4 °C) (11). Our original IRMA was performed at room temperature. Spontaneous activation of prorenin over time has been detected at room temperature, but is virtually absent at 37 °C(12). We therefore attempted to prevent the inadvertent activation of prorenin by shortening the incubation time of the assay and raising the incubation temperature to 37 °C.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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avidin-coated plastic beads, washing buffer, and sheep serum
Biotinylated bovine serum albumin was coupled to 8-mm diameter
polystyrene beads (Precision Plastic Balls), after which the beads were
coated with avidin (15) and stored at 4 °C. The washing
buffer, phosphate-buffered saline containing 0.1 mL/L Triton
X-100, was provided by Nichols Diagnostics. Serum from healthy
sheep was heat inactivated for 1 h at 56 °C.
renin calibrators
Recombinant human renin was provided by Nichols Diagnostics. We
calibrated this renin preparation against the WHO human kidney renin
standard, International Reference Preparation (IRP), lot no. 68/356
(National Institute for Biologic Standards and Control, Potters Bar,
Hertfordshire, UK) (16) in the enzyme-kinetic PRC assay (see
below). Serial dilutions of human recombinant renin to be used for
constructing the calibration curve were made up in undiluted sheep
serum. The sheep serum content of the renin calibrators was, therefore,
near 100%, contrary to what was stated in our previous report(7) on a renin IRMA. The dilutions of the renin calibrators
were then lyophilized and stored at 4 °C. Before assay, the
lyophilisates were reconstituted in distilled water.
renin inhibitor
Remikiren, an active site-directed renin inhibitor, was obtained
from Hoffmann-La Roche (17). Remikiren is a nonpeptide
transition-state analog with a Mr of
726. The IC50 for purified human renin is
0.7 x 10-9 mol/L, and the
Ki for the reaction with human renin
is 3 x 10-10 mol/L (17). Only
freshly prepared solutions of remikiren were used.
angiotensinogen
Angiotensinogen was prepared from the plasma of nephrectomized
sheep (18). The partially purified preparation was dialyzed
against 0.15 mol/L phosphate-buffered saline, pH 7.4, and
neomycin sulfate was added (final concentration, 2 g/L). The final
preparation had an angiotensinogen concentration of 1.2 x
10-6 mol/L, determined as described below.
nonproteolytic activation of prorenin
Prorenin was treated with the renin inhibitor remikiren to change
the molecular conformation from an inactive form into a form that is
recognized by the mAb specific for renin (19). We refer to
this remikiren-induced conformational change of prorenin as
"activation", despite the fact that the prorenin-remikiren complex
lacks enzymatic activity. To activate all the prorenin in plasma, we
added 1 volume of remikiren (10-3 mol/L) to 10
volumes of plasma and incubated the mixture for 24 h at 4 °C(19).
assays
Enzyme-kinetic assays of renin.
To measure PRC, we used our
in-house assay, as described previously(20)(21). In short, we added 250-µL aliquots
of sheep angiotensinogen solution to 25- to 250-µL aliquots of plasma
and adjusted the volume to 500 µL with 0.15 mol/L phosphate-buffered
saline, pH 7.4, and added 17.5 µL of a protease inhibitor solution
consisting of two volumes of 0.34 mol/L 8-hydroxyquinoline sulfate, one
volume of 0.287 mol/L phenylmethylsulfonyl fluoride in ethanol, two
volumes of 0.5 mol/L disodium EDTA, and two volumes of aprotinin
(10 000 kallikrein-inhibiting units/mL). We incubated the mixture at
37 °C for at least two different time periods between 0.25 and
3 h to verify linear Ang I generation. The final concentration of
sheep angiotensinogen in the incubation mixture was 6 x
10-7 mol/L (Km,
2 x 10 -7 mol/L). Only incubations in which <5% of
the angiotensinogen was hydrolyzed were accepted for calculating the
concentration of naturally occurring renin. Parallel incubations at
0 °C served as blanks. The generated Ang I was measured by RIA(22). Results of the PRC measurements were expressed as
mU/L, as calibrated with the international renin standard. Under the
conditions of the assay, 1 mU of renin generated 163 pmol (212 ng) of
Ang I per hour. The lower limit of detection was 1 mU/L.
For the PRA assay, we used a modification of the method proposed by Sealey (23). The method was modified to include blank subtraction, and the 18-h incubation for low renin samples was eliminated. In this assay, 50 µL of maleic acid, pH 5.7, and 12.5 µL of a protease inhibitor solution consisting of one volume of 0.287 mol/L phenylmethylsulfonyl fluoride in ethanol, two volumes of 0.5 mol/L disodium EDTA, and two volumes of 100 g/L neomycin sulfate were added to 0.5 mL of plasma. The mixture was then incubated at 37 °C for at least two time periods between 0.5 and 3 h to check for linear generation of Ang I. The generated Ang I was quantified by RIA (22). Results were expressed as nmol Ang I/L per hour. The lower limit of detection was 0.08 nmol Ang I/L per hour.
Assay of angiotensinogen.
The concentration of angiotensinogen
in plasma was determined as the maximal quantity of Ang I that was
generated during incubation at pH 7.4 and 37 °C of plasma to which
recombinant human renin had been added at a final activity of 20 000
mU/L. The results were expressed as nmol/L (21).
IRMAs.
To 200-µL aliquots of untreated plasma,
remikiren-treated plasma, or renin calibrators, we added 100 µL of a
1:1 mixture, by volume, of biotinylated mAb R3-36-16 (0.5 mg/L) and
radiolabeled [~250 000 counts per minute (cpm)] mAb R1-20-5. We
incubated this mixture with a polystyrene, avidin-coated bead in
duplicate for either 24 h at room temperature (IRMA 22 °C,
24 h) or for 6 h at 37 °C (IRMA 37 °C, 6 h). After
incubation, we washed the beads three times and then transferred the
washed beads to clean tubes. The radioactivity of the bound antibody
was counted for 5 min in a gamma counter. The assay measures renin in
the untreated plasmas and renin plus prorenin in the remikiren-treated
plasmas. The difference between the results of the two measurements was
the prorenin.
To test our hypothesis that comeasurement of prorenin as renin was caused by a conformational change in the prorenin, we also performed another, two-step sandwich assay. This assay was a modification of an assay we described earlier (14) and measured prorenin directly after remikiren activation. In this assay, an avidin-coated bead carrying 1 µg of biotinylated F258-37-B1 was incubated for 6 h at 37 °C or for 24 h at 22 °C with plasma from a patient with high plasma prorenin (prorenin, 2757 mU/L; renin, 53.1 mU/L). The F258-37-B1-coated bead bound the exposed propeptide of the active form of prorenin (14), but did not bind renin or native, inactive prorenin. The half-time of binding of active prorenin to the bead at 37 °C was ~12 min. After incubation, we rinsed the bead three times with 2 mL of phosphate-buffered saline containing 1 g/L bovine serum albumin to remove all renin and inactive prorenin. We then incubated the bead with sheep serum containing remikiren (10-4 mol/L) and radiolabeled R1-20-5 for 24 h at 22 °C. The tracer antibody bound the active prorenin trapped on the bead. We added remikiren in this second step to ensure that any active prorenin captured on the bead in the first step was kept in its active conformation. After incubation, we washed the beads with 3 x 2 mL of washing buffer and counted bound radioactivity after the bead was transferred to a clean tube. A standard line of remikiren-treated recombinant human prorenin in sheep serum was used (14). The lower limit of detection of this assay was ~10 mU/L active prorenin. The interassay CV at <100 mU/L active prorenin was ~10%.
collection of plasma samples
Plasma samples were from healthy subjects, patients
with essential hypertension either not receiving therapy or receiving
enalapril, patients with renovascular hypertension either not receiving
therapy or receiving enalapril, patients with hepatic cirrhosis, and
patients with primary aldosteronism (Conn syndrome). Individuals
with conditions where a high prorenin-to-renin ratio is not unusual,
i.e., pregnant women, women with ovarian hyperstimulation, women with
preeclampsia, women taking oral contraceptives, and patients with
diabetes mellitus, were also studied. Blood sampling was performed
according to our in-house protocol. Blood samples were collected from
subjects after cannulation of an antecubital vein and 30–45 min of
supine rest. The samples were collected into tubes containing 0.2 mL of
0.646 mol/L citrate per 10 mL of blood and centrifuged immediately at
room temperature. The plasma was stored at -20 °C. Shortly before
each assay, plasma samples were thawed rapidly and kept at room
temperature.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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depicts a comparison of the calibration curve of IRMA 37 °C,
6 h with the calibration curves of IRMA 37 °C, 24 h and
IRMA 22 °C, 24 h. The results, expressed as cpm, were linear
over a wide range of renin concentrations. IRMA 37 °C, 24 h and
IRMA 22 °C, 24 h produced identical results. It appears,
therefore, that the reagents were stable at 37 °C. The calibration
curve of IRMA 37 °C, 6 h was parallel to those of the 24-h
IRMAs, but the values were 25% lower. This difference was fully
explained by the first-order rate constant for the binding of the
radiolabeled mAb to renin at 37 °C (0.23
h-1; data not shown). For practical purposes, we
chose IRMA 37 °C, 6 h instead of IRMA 37 °C, 24 h as
our routine assay, although the quantity of radiolabeled mAb
trapped by renin was somewhat lower after 6 h than after 24
h.
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We tested the linearity of the assay by preparing dilutions of plasma
samples with 5.7–2106 mU/L renin. Dilutions were made up in the same
matrix, i.e., sheep serum, that was used for the calibration curve.
Plots of the results, expressed as mU/L, against the dilution factor
gave straight lines with the correct slope (Fig. 2
).
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Analytical recovery.
The human kidney renin IRP was added to
plasma samples containing 7.4–155 mU/L renin. As shown in Table 1
, IRMA 37 °C, 6 h demonstrates excellent analytical
recovery of both low and high quantities of added renin. The mAbs we
used are capable of inhibiting the Ang I-generating activity of renin(13). To investigate whether angiotensinogen might interfere
with the binding of renin to these antibodies, we added the human
kidney renin IRP to a plasma obtained from a pregnant woman. This
plasma (plasma D in Table 1
) had an angiotensinogen concentration of
3208 nmol/L, which is approximately threefold higher than the reference
value. Angiotensinogen at this high concentration did not influence the
results of IRMA 37 °C, 6 h (see Table 1
).
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Assay imprecision and detection limits.
As shown in
Fig. 1
, the detection limit of IRMA 37 °C, 6 h, defined as 3 SD
above the zero calibrator, was 1.3 mU/L. The radioactivity of the zero
calibrator of this calibration curve was ~150 cpm, and that of the 4
mU/L calibrator was ~300 cpm. Thus, the result for the calibrator was
close to the blank. This raised concern about the reliability of the
IRMA renin measurements at the lower end of the concentration range.
This issue is addressed in Fig. 3
, which shows the results, expressed as cpm, for 4 mU/L human
recombinant renin and for nonspecific binding (blank) in 32 individual
assays. Fig. 3
shows adequate separation between the 4 mU/L calibrator
and the blank in each of the 32 assays. The variability of the
duplicate blanks or the duplicate 4 mU/L calibrators was low. The
difference between the duplicate blanks, given as median (range), was
13.5 cpm (1–38 cpm), or 11% (1–35%) of the mean blank (n =
32). The difference between the duplicate 4 mU/L calibrators was 14 cpm
(1–32 cpm), or 6% (0–15%) of specific binding.
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The intra- and interassay CVs for IRMA 37 °C, 6 h; IRMA
22 °C, 24 h; and the enzyme-kinetic PRC assay at low, medium,
and high renin activities in plasma are compared in Table 2
. The intraassay CVs were similar for the three assays.
The interassay CVs for the plasmas with low renin activity were,
however, higher for the IRMAs than for the enzyme-kinetic PRC assay.
The "functional sensitivity", i.e., the minimum renin activity that
could be measured from assay to assay with <20% CV, was ~4 mU/L
(see Table 2
).
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comeasurement of prorenin
The results for a plasma pool from healthy subjects measured with
IRMA 37 °C, 6 h; IRMA 22 °C, 24 h; and the
enzyme-kinetic PRC assay are compared in Table 3
. The renin activity in the plasma pool measured with IRMA
37 °C, 6 h was equal to that measured with the enzyme-kinetic
PRC assay and was ~25% lower than the activity measured
with IRMA 22 °C, 24 h. In contrast, the results obtained with
IRMA 37 °C, 6 h and with the enzyme-kinetic PRC assay in a
dilution of the human kidney renin IRP in sheep serum were not
different from the results obtained with IRMA 22 °C, 24 h. This
is an indication that prorenin interferes with the measurement of renin
in IRMA 22 °C, 24 h. To further demonstrate that a
conformational change in prorenin at 22 °C is the source of renin
overestimation, we also used a two-step IRMA (at 22 and 37 °C) that
measures the active conformation of prorenin. When the first step of
the assay incubation was performed at 37 °C for 6 h, very
little active prorenin was measured in the second step (18 mU/L, <1%
of total prorenin and close to the detection limit of the assay). In
contrast, when the same plasma was incubated with the same beads at
22 °C for 24 h, the active prorenin measured in the second step
was 149 mU/L, i.e., 5.4% of total prorenin. This indicates that in the
course of incubation, prorenin is activated and then captured by the
F258-37-B1-coated bead at 22 °C, but not at 37 °C. The results
were the same when the assay was repeated with the same plasma, and
similar results were observed for three other plasmas with high
prorenin. Activation of prorenin at 22 °C, but not at
37 °C, also explains the results presented in Fig. 4
, in which we compare the results obtained with IRMA 22 °C,
24 h and IRMA 37 °C, 6 h with the results obtained with
the enzyme-kinetic PRC assay for plasmas having different
prorenin-to-renin ratios. Overestimation of renin was frequent in IRMA
22 °C, 24 h, and was most pronounced at high prorenin-to-renin
ratios (Fig. 4
, left-hand plot). Overestimation of renin was not seen
in IRMA 37 °C, 6 h. Fig. 5
shows a Bland-Altman plot comparing IRMA 37 °C, 6 h
with the enzyme-kinetic PRC assay. Because the renin activity ranged
over three orders of magnitude and the differences were proportional to
the activity, the mean of the two assays was log-transformed and the
difference was corrected for the renin activity. The plot demonstrates
the good agreement between IRMA 37 °C, 6 h and the
enzyme-kinetic PRC assay for the entire range of renin activity in
plasma.
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In Table 4
, the results presented in Figs. 4
and 5
are grouped according
to diagnostic categories. Table 4
also compares the three types of
renin measurement (IRMA 22 °C, 24 h; IRMA 37 °C, 6 h;
and enzyme-kinetic PRC assay) with the measurement of PRA in different
patient groups. The percentage of overestimation that was seen in IRMA
22 °C, 24 h was most pronounced in patients with diabetes
mellitus, in pregnant women with or without preeclampsia, and in
gonadotropin-treated women. These were the patient groups with the
highest prorenin-to-renin ratio. IRMA 22 °C, 24 h did not
overestimate renin in patients with renovascular hypertension treated
with enalapril and patients with liver cirrhosis. In these patient
groups, the prorenin-to-renin ratio was abnormally low.
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Many laboratories measure PRA instead of renin concentration. The
results of the PRA assay are determined not only by the concentration
of renin, but also by the concentration of angiotensinogen in plasma.
We compared the results obtained with IRMA 37 °C, 6 h with
those obtained with the PRA assay. Fig. 6
shows the results for healthy subjects, for patients with
untreated essential hypertension, and for patients with Conn syndrome.
The angiotensinogen concentration in these plasma was normal, and the
renin was normal or low. The results of the PRA assay (with nmol Ang I
· L-1 · h-1 as the units) were 0.07 times the
values found with the IRMA 37 °C, 6 h (with mU/L as the units). A
similar correlation was seen in other patient groups having normal
renin and angiotensinogen concentrations in their plasma (see Table 4
).
As expected, the PRA-to-IRMA ratio was higher in patients with
abnormally high angiotensinogen, and the ratio was lower in patients
with abnormally low angiotensinogen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that this problem is solved in the improved assay. The tertiary structure of renin and prorenin has the form of two lobes separated by a cleft that contains the enzyme's active site (24)(25). In enzymatically inactive intact prorenin, the propeptide is folded in this cleft, and angiotensinogen cannot reach the active site. This so-called closed form of prorenin is in dynamic equilibrium with an open form in which the propeptide, although still covalently linked with the renin portion of the molecule, has been displaced from the cleft, and angiotensinogen can now reach the active site (14). The transition of the closed form into the open, active form takes hours, and the equilibrium is shifted toward the open form by lower temperatures (cryoactivation)(12) or by the addition of active site-directed renin inhibitors (19). Our results, especially those of the two-step IRMA that captures active prorenin, demonstrate that the comeasurement of prorenin we observed in the original IRMA 22 °C, 24 h, is caused by the formation of open prorenin. This problem is almost completely overcome in IRMA 37 °C, 6 h.
IRMAs usually are performed at room temperature. One may therefore wonder whether the reagents we used are stable at 37 °C. Our observation that the calibration curves for IRMA 37 °C, 24 h and IRMA 22 °C, 24 h were identical strongly suggests that the reagents are stable at 37 °C.
The renin calibrator was diluted in sheep serum to minimize nonspecific binding of the mAbs. Theoretically, the binding characteristics in the sheep serum matrix might be different from those in human plasma. The recovery of renin calibrator added to human plasma was close to 100%, and serial dilutions of human plasma produced decrements of binding that were linearly correlated with the dilution factor. The close agreement between the results of IRMA 37 °C, 6 h with those of the enzyme-kinetic PRC assay also indicates that the difference in the reaction milieu between plasma samples and calibrators did not affect the results.
For plasma samples with normal or increased renin, IRMA 37 °C, 6 h was as precise as the enzyme-kinetic PRC assay (interassay CV <10%). For plasma samples with low renin activity (<10 mU/L), the precision of IRMA 37 °C, 6 h was less than that of the enzyme-kinetic PRC assay. The functional sensitivity, defined as the minimum activity that could be measured from assay to assay with <20% CV, was ~4.0 mU/L. Our results show this to be sufficient for measuring renin in plasmas from patients with suppressed renin, such as patients with Conn syndrome or low-renin essential hypertension.
Many laboratories still use the enzyme-kinetic PRA assay. From our results, it appears that the patients who were diagnosed as having a low plasma renin activity on the basis of results obtained with IRMA 37 °C, 6 h also had abnormally low PRA.
The improved IRMA is specific for renin and appears to be a simple, precise, and accurate method for measurements in clinical plasma samples. This assay is easier to standardize than the enzyme-kinetic assays that rely on endogenous angiotensinogen, such as the PRA assay. The results obtained with the improved IRMA are expressed in terms of the internationally recognized human renin standard and, therefore, permit ready comparison between different laboratories.
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Acknowledgments |
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Footnotes |
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References |
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), a patient with
untreated renal artery stenosis (•), a pregnant woman (
), a
patient with low-renin hypertension (
), and a patient with Conn
syndrome (
).
), patients with essential hypertension (•),
and control subjects (
).