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Technical Briefs |
1 Department of Internal Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
aauthor for correspondence: fax 31-0-10-4634531, e-mail boomsma{at}inw1.azr.nl
Measurements of the plasma concentrations of one or more of the natriuretic peptides can be a valuable tool in the diagnosis, prognosis, and follow-up of patients with cardiac dysfunction (1)(2). Measurement techniques have also improved, allowing for less time-consuming direct assays than previous methods, which often required the prior extraction of peptides from plasma. However, improvement is still needed (3).
In most research settings, blood sampling is performed under carefully controlled conditions, involving the introduction of a catheter and ample bed rest before the collection of blood. Such procedures are necessary for the reliable interpretation of many neurohormone measurements. Catecholamines, for example, can fluctuate widely depending on the blood-sampling procedure and posture. Because strictly adhering to rigorous blood-sampling protocols is often impossible in everyday clinical situations, we investigated the variation in measured natriuretic peptide concentrations because of differences in blood-sampling conditions.
We have measured atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), N-terminal-pro-ANP (Nt-ANP), as well as the catecholamines noradrenaline (NOR), adrenaline, renin (REN), and aldosterone (ALDO). We studied subjects in the relevant age range (>40 years) with diseases of interest in four different blood-sampling conditions, while continuing appropriate medications for these conditions. Twenty-three subjects (55 ± 10 years; range 42–74 years) participated in the study after giving their informed consent. Six subjects were apparently healthy controls (46 ± 5 years; two females), nine were hypertensive patients (53 ± 7 years; two females), and eight (64 ± 9 years; three females) were patients with diseases known to lead to increased concentrations of natriuretic peptides: congestive heart failure (four subjects) and chronic renal failure (four subjects). All patients were taking appropriate medications for their conditions. Immediately after entering the examination room in our department, subjects sat down and a small catheter was inserted into an antecubital vein. Blood was sampled directly and then after 30 min of continued quiet sitting. Blood was sampled again both after 30 min of lying on a bed (bed rest) and after 30 min of standing and walking. The whole procedure was repeated on a second visit, at least 7 days later (median interval, 12 days) to assess intraindividual variation from one day to another. The procedure was always performed in the morning between 0900 and 1200.
Blood was collected in two different tubes: (a) one tube containing EDTA and aprotinin (1.9 mg and 100 kIU/mL of blood, respectively) for measurement of natriuretic peptides and (b) one heparin-containing tube containing 1.2 mg of glutathione/mL of blood for measurement of NOR, adrenaline, REN, and ALDO. The tubes were centrifuged (3000g) within 30 min at 4 °C for 10 min, and the plasma was separated and stored at -70 °C until assayed. ANP was determined by RIAs (Nichols Institute) after Sep-Pak C18 extraction from plasma. BNP was measured with immunoradiometric assays from Shionoria, and Nt-ANP was measured with RIAs from Biotop, both without extraction. Catecholamines were measured by HPLC with fluorometric detection after extraction from plasma and derivatization with dimedone (4). ALDO was determined with RIAs from Diagnostic Products Corporation. REN was determined by RIA of generated angiotensin I (5). All methods used had intraassay CVs in the relevant range of <10%. All samples from one subject were measured in the same assay.
Mean neurohormone concentrations of samples collected after 30 min of
bed rest on the first visit are reported in Table 1
, as are the concentrations for samples collected directly after
the subjects arrived and the samples collected after 30 min of sitting
and after 30 min of standing and walking. The concentrations in Table 1
are also expressed as percentages of the individual neurohormone
concentrations after 30 min of bed rest. ANOVA analysis showed that the
difference between the four procedures is significant for all
neurohormones, but posttests with Bonferroni correction indicated that
the differences in the natriuretic peptides were small and often (with
BNP) not significant. In contrast to the well-known fact that REN and
ALDO vary quite considerably under different blood-sampling conditions,
natriuretic peptides appeared to be much less variable. Samples
collected directly after patients arrived gave somewhat higher values,
but values obtained during quiet sitting were indistinguishable from
those obtained during bed rest. For Nt-ANP, the differences were the
smallest, which was not surprising in view of its relatively long
half-life. These findings agree with the results of Wijbenga et al.
(6), who, in a study on the response of natriuretic peptides
to exercise, found the highest percentage of change in ANP, a smaller
percentage of change in BNP, and the smallest percentage of change in
Nt-ANP. Nt-BNP, which was measured in that study (6) but not
in our present study, changed more than Nt-ANP but less than BNP. For
the catecholamines REN and ALDO, there were considerable differences in
the values obtained directly after arrival and after 30 min of bed
rest, but the differences between the values obtained after 30 min of
sitting and after 30 min of bed rest were much smaller. Furthermore,
Bonferroni posttests produced statistically significant results for
ALDO only. The lack of statistical significance for the difference
between sitting and bed rest values for NOR and adrenaline contrasts
with previous findings from our laboratory (7). In that
study, however, only young healthy subjects took part, whereas in the
present study, subjects were older, and most had diseases associated
with an activation of the sympathetic nervous system. We should mention
that in younger subjects, some neurohormones may have a greater
response than in the group of older subjects we studied. The effects of
medication used by all patients may have also played a role.
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At the second visit (again as a percentage relative to the samples
collected at the first visit after 30 min of bed rest), the pattern was
much the same as at the first visit. The difference between the four
procedures was significant for all neurohormones, and posttests broadly
showed similar results as on the first visit. Correlations between
absolute values for the same blood-sampling procedure at the first
visit vs those at the second visit were all significant, except for NOR
after 30 min of standing and walking. A good impression of individual
differences could be obtained by constructing Bland–Altman plots
(8), which are shown for the natriuretic peptides in the
sitting situation in Fig. 1
. These indicate that although usually there was good agreement
between measurements on the two different visits, occasionally a large
difference was found. This could be a result of the timing of the
medication used (e.g., the effects of angiotensin-converting enzyme
inhibitors on REN and ALDO values), but sometimes also for no apparent
reason. Here again, the differences in Nt-ANP and BNP appeared to be
the smallest.
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Which of the various natriuretic peptides is (are) most informative with regard to the diagnosis, course, and treatment of heart failure is still being debated. From the viewpoint of stability in blood samples, the N-terminal peptides, as well as BNP, appear to be the best (9)(10)(11)(12)(13). ANP is less stable, although not as unstable as has been suggested (14). In addition, because of longer half-lives, short-term fluctuations will have less of an effect on the measured concentrations of the N-terminal peptides than of the C-terminal peptides. However, clinical studies are needed to determine which is (are) the most informative.
We conclude that the measurement of natriuretic peptides in a single blood sample, obtained after 30 min of quiet sitting, can give reliable and reproducible basal values. Measurements of the catecholamines REN and ALDO in the same blood sample are only marginally higher than values obtained after 30 min of bed rest. Especially for BNP and Nt-ANP, blood sampling directly after arrival may be a reasonable alternative when time and/or facilities are limited.
References
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