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Technical Briefs |
BCO Analytical Services, PO Box 2176, 4800 CD, Breda, The Netherlands
a author for correspondence: fax +31 765 737777, e-mail info{at}BCO.NL
Renin (EC 3.4.23.15) catalyzes the proteolytic degradation of angiotensinogen to angiotensin-I. Plasma renin activity can be assessed by detection of the amount of angiotensin-I produced under well-defined incubation conditions. Despite numerous studies of incubation conditions (1)(2)(3)(4)(5)(6)(7)(8), the temperature optimum of the renin enzyme activity and the effect of the assay temperature on the reproducibility of renin activity measurement have not been reported. Here we draw attention to the temperature effect on the human plasma renin activity over the range between 30 and 50 °C.
Blood samples from two men, coded A and B, ages 34 and 40 years, were collected in 10-mL EDTA-containing evacuated polyester tubes (Venoject II, type VP-100DK; Omnilabo Nederland, Breda, The Netherlands) and mixed for 1 min. Within 10 min of collection, each sample was centrifuged at ambient temperature for 15 min at 1300g. The plasma obtained was separated from the blood cells within 10 min thereafter and, with the use of polyethylene pipettes, was transferred into polystyrene tubes in small aliquots. Also used were an EDTA-anticoagulated plasma selected without conscious bias from leftover laboratory ("waste") samples (coded C) and a RIANEN Renin kit (see below) control sample (coded D). Subjects' samples were stored at ~-18 °C until analysis; the kit control sample was reconstituted with ice-cold distilled water; aliquots were stored in polystyrene tubes as instructed in the package insert. Samples were thawed at room temperature within 1 h of assay.
Renin activity was measured with the RIANEN (NEA-104, 105; Du Pont NEN Research Products, Boston, MA) test kit, including the renin kit control. Reconstitution and dilution was performed with doubly distilled, pyrogen-free water (B. Braun NPBI, Oss, The Netherlands). Into noncoated tubes kept on melting ice were added consecutively 500 µL of sample, 10 µL of dimercaptol solution, 10 µL of 8-hydroxyquinoline solution, and 1 mL of maleate buffer, pH 6.0. After mixing, we obtained split samples for incubation at two temperatures by transferring 1.0 mL of each sample into a second tube. The first series was incubated at ~4 °C for 60 min; the second series was incubated for the same period at a fixed temperature (±0.3 °C) selected from the range of 30–50 °C. After this incubation period, all samples were put on melting ice. From each, 100 µL was transferred into each of two duplicate tubes, followed by addition of 100 µL of 125I-labeled angiotensin-I solution and 100 µL of antiserum solution, and mixed thoroughly. Similarly, 100 µL of calibrators (in duplicate) were mixed with 100 µL of 125I-labeled angiotensin-I solution and 100 µL of antiserum solution. After incubation for 2 h at ambient temperature, 500 µL of second antibody solution was added to each sample tube and mixed thoroughly. After another incubation for 20–30 min at ambient temperature, the samples were centrifuged at 1000g for 10 min and aspirated. Finally, the 125I radioactivity remaining in the tubes was counted with an automated gamma counter (LKB Wallac Gammamaster; Wallac E&EG, Breda, The Netherlands). RIACALC software (LM Software, Wallac Oy, Finland) was used for data handling. Renin activities in samples A and B were assessed several months before those in samples C and D.
Dependence of renin activities (expressed in nanograms of angiotensin-I
per milliliter of plasma per hour) on the temperature increase are
presented in Fig. 1
. The results in samples A and B revealed that the renin
activity increased markedly with the temperature in the range
34–40 °C. Additional analysis of the two extra samples (human
plasma sample C and a control sample D) showed maximum human plasma
renin activity at ~45 °C. The temperature-dependent renin activity
course could be an effect of the pH change during the incubation as a
result of the temperature increase. Measurement in two samples of the
effect of the temperature increase on pH at three different
temperatures (25, 37, and 45 °C) detected no significant change of
the pH (not shown). On the other hand, a temperature optimum could
be artificially created, if at a temperature >45 °C the angiotensin
were broken down by increased activities of proteolytic enzymes present
in the samples. To exclude such phenomena, we assayed two samples
enriched with exogenous angiotensin-I (final concentration in the
samples: 2.1 µg/L) and incubated for 1 h at 37 or 45 °C. From
the measurements of angiotensin-I, the recovery of the added
angiotensin-I was calculated. The mean recovery at 37 °C was 95%
and 93% of that at 45 °C, suggesting that the optimum temperature
of the renin activity at nearly 45 °C is not caused by a breakdown
of angiotensin-I, e.g., induced by activation of proteolytic enzymes in
the samples.
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Some investigators (9)(10)(11)(12) prefer to assess renin concentrations by mass measurement with immunochemical methods rather than measure renin activity, because the immunochemically determined concentration will not be influenced by physiological or pathological variations of the renin substrate, angiotensinogen (9)(10). In favor of mass measurement of the renin concentration is the minimal risk of generating different amounts of angiotensin-I from identical plasmas investigated in different laboratories (11)(12). However, the biological activity of renin is of clinical relevance (13), and mass measurement with immunochemical methods such as the IRMA described recently (14) is subject to cross-reactivity of the antibodies used, which can lead to overestimation of renin in, e.g., plasma of patients with relatively high concentrations of prorenin (13). Rather, the generation of different amounts of angiotensin-I in identical plasmas determined in different laboratories using activity measurements (11) may be explained by the lack of an assessed temperature optimum or a recommended temperature.
Because incubation temperatures should be selected to give optimum sensitivity and minimum analytical variation, we recommend that plasma renin activity be measured at 45 °C.
References
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, plasma B; 
, plasma C; stars, kit
control (D).