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1Department of Anesthesiology and 2Institute of Physiology, University of Lübeck, D-23538 Lübeck, Germany; 3Institute of Clinical Biochemistry, Rikshospitalet, University of Oslo, NO-0316 Oslo, Norway; and 4Immundiagnostik AG, D-64625 Bensheim, Germany
Submitted 10 November 2003 ; accepted in final form 25 February 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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intrathoracic blood volume; fluid homeostasis; posture; cardiorenal integration; natriuretic peptides
These findings are suggestive that RLX may play a role in cardiorenal integration and that the plasma levels of this hormone may be related to changes in cardiac filling pressures and are possibly linked to the natriuretic peptide system. No data are available that assess whether the plasma levels of RLX in healthy individuals are influenced by postural changes and acute fluid loading as factors that are known to modify natriuretic peptide release (10, 20).
Hence we hypothesized that changes in thoracic blood volume (TBV), induced either by tilting or by intravenous volume loading, might induce concomitant changes in plasma and urinary RLX levels in healthy male volunteers. The activity of the natriuretic peptide system in blood and urine was determined by analyzing the course of the NH2-terminal prohormones of ANP (NT-pro-ANP) (21) and brain natriuretic peptide (BNP; NT-pro-BNP) (16). Additionally, urinary excretion of urodilatin (Uro; UUro), a kidney-derived member of the natriuretic peptide family system, was studied (8).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Tilting Protocol
Sixteen volunteers were randomly divided into two groups of n = 8 and studied in different body positions for 2 h each. After a resting period in the supine position, subjects were either tilted to a 15° head-down position or to the feet-down position, were brought back into the supine position, and afterward were tilted into the opposite direction.
Hemodynamics [heart rate (HR), electrocardiogram, mean arterial blood pressure, automated oscillometric sphygmomanometer] were recorded every 15 min and averaged for respective time periods. The end-systolic left atrial diameter (LAD) was determined by transthoracic echocardiography (ATL Ultrasound, Apogee; CX 100-150) in the long parasternal view. LAD measurements were performed after 1 h in the respective body position and are given as the mean of three measurements. Blood for determination of blood chemistry and hormones was sampled every hour, beginning at 0900.
Volume-loading Protocol
Ten volunteers were randomly studied during an observation period of 10 h (control group) in the supine position or were subjected to an intravenous infusion of 15 ml/kg of 0.9% NaCl applied during 60 min with an infusion pump, from 1000 to 1100 (volume group). Studies were performed on different days at least 2 wk apart.
In both groups, blood sampling for determination of blood chemistry and hormones, as well as echocardiographic determinations of LAD, was performed at 0900, 1000, 1100, 1200, 1400, 1600, and 1800. In the volume group, urine was sampled by spontaneous voiding at 0800, 1000, 1100, 1200, 1400, 1600, and 1800. Hemodynamics were determined every 15 min and averaged for respective time periods.
No urine sampling was performed in the control group, as we have repeatedly had the experience (unpublished observations) that it is often impossible to get precise and frequent urinary samples from volunteers without the concomitant application of fluid. Because water diuresis has been shown to induce a counterregulatory decrease in renal natriuretic peptide excretion (9), we deemed it more appropriate to waive urine samples in the control group instead of water loading all volunteers with the possible risk of interference with hormonal regulation.
Blood and Urine Analyses
Blood for hormone analysis was sampled in EDTA tubes containing 5,000 units aprotinine (Trasylol, Bayer, Germany) and lithium-heparin tubes (for clinical chemistry), as appropriate. Blood and urine samples were immediately spun for 10 min at 3,400 rpm at 4°C. Supernatants were stored at –80°C until analysis. Plasma and urine electrolytes (flame photometrically) and creatinine (enzymologically) were determined on standard hospital analyzers.
Renal functional parameters [fractional excretion of sodium (FENa) and creatinine clearance] were calculated according to standard formula. Urinary excretion of hormones was expressed as the ratio between the hormone and the creatinine concentration.
Determination of RLX
RLX was determined by a sandwich-type enzyme immunoassay, as described previously (1, 5). In short, this assay is based on two specific polyclonal antibody raises in rabbits against recombinant human gene H2-RLX. The detection limit of this assay is 1.2 pg/ml. The standard range is 1.2–250 pg/ml. Recovery in plasma is 97 ± 4.5%. The intra-assay coefficient of variance at 15.3 pg/ml was 9.6% (n = 18); the interassay coefficient of variance at 15.6 pg/ml was 10.2% (n = 12). The assay has a cross-reactivity of <0.01% with insulin, insulin-like growth factor, LH, and FSH.
Determination of NT-pro-ANP
NT-pro-ANP was determined by a competitive-binding radioimmunoassay with magnetic solid-phase technique, in a modification of Sundsfjord et al. (21), by using the same rabbit anti-rat pro-ANP polyclonal serum, human pro-ANP-(1–30) from Peninsula Laboratories (Bachem, St. Helene, UK) as the standard, and iodined pro-ANP(1–30) purified by HPLC for radiolabeling. To achive high sensitivity and good precision, Dynabeads M280 with sheep anti-rabbit IgG (Dynal Biotech, Oslo, Norway) as solid-phase and second antibody were used. The coefficients of variance at 425, 1,163, and 2,490 pmol/l were 7.5, 3.7, and 3.4%, respectively. The detection limit was 30 pmol/l.
Determination of NT-pro-BNP
NT-pro-BNP was determined by an electrochemoluminescence immunoassay (Elecsys pro-BNP sandwich immunoassay; Roche Diagnostics, Basel, Switzerland) on Elecsys 2010 (16). The mean intra-assay variance was 4.3% (range: 2.7–5.9% for plasma samples with a concentration between 7.6 and 2,732 pmol/l, with an interassay variance of 3.2%). The lower detection limit was 0.6 pmol/l.
Determination of Uro
Uro was determined by a RIA for human Uro (Immundiagnostik, Bensheim, Germany), as described previously in detail (15). The antiserum does not cross-react with ANP-(99–126), NT-pro-BNP, or BNP. The sensitivity of the method was 10.5 pg/ml, and the intra- and interassay variabilities were 8.1 and 9.7%, respectively.
Statistical Analyses
Data in the text are given as means ± SD, data in Figs. 1– 4 are given as means ± SE. Significance was set to P < 0.05.
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Volume-loading protocol. Due to the small sample size, calculations on the raw data were performed nonparametrically. Intraindividual differences between the control and the volume group were analyzed by Friedman's test followed by Wilcoxon's matched-pairs test. Bonferroni correction was not used. Intraindividual differences during the observation period were determined by Wilxoxon's matched-pairs test. Spearman's rank correlation test was used for correlation analyses.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Hemodynamics. Neither HR nor arterial blood pressure showed significant intra- or interindividual changes throughout the observation period (Table 1). A significant difference in LAD was observed during the second tilting period (Table 1).
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Plasma hormone levels. The course of normalized NT-pro-ANP, NT-pro-BNP, and RLX levels is depicted in Fig. 1. NT-pro-ANP levels were higher during HD than during FD in the second tilting period. NT-pro-BNP levels increased over time until 1500 but were not different between both groups. No significant interindividual variations were observed in plasma RLX levels. The intraindividual course of this hormone was comparable in both groups, showing a surge in normalized RLX levels at 1500 compared with baseline and an increase from 1500 to 1600 back to baseline levels.
Volume-loading Protocol
Hemodynamics. No significant within- and between-group differences were observed in HR and mean arterial blood pressure (Table 2). A small, albeit significant, increase in LAD was observed after the saline infusion from 1100 to 1200. Thereafter, LAD decreased back to baseline levels (Table 2).
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Plasma hormone levels. The plasma levels of NT-pro-ANP, NT-pro-BNP, and RLX are depicted in Fig. 2. In the volume-loading group, NT-pro-ANP levels increased immediately with a peak 2 h after the infusion. A moderate increase was also observed in the control group. However, after 1200, NT-pro-ANP levels in this group were not different from baseline levels. NT-pro-BNP showed a delayed increase up to the end of the observation period in both groups. NT-pro-BNP levels in the volume-loading group were significantly higher than in the control group after 1300. No significant variations or between-group differences in RLX levels were observed.
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Correlation analyses. Correlation analysis of plasma hormone levels at baseline between the control and the volume-loading group for determination of the reliability of the measurements revealed the following significant correlations: NT-pro-ANP, rho = 0.91; NT-pro-BNP, rho = 0.82; and RLX, rho = 0.91.
Correlation analyses between urinary functional parameters and hormonal plasma levels and urinary excretion of these hormones are given in Table 3. These analyses revealed minor relationships between plasma levels of NT-pro-ANP and NT-pro-BNP on one hand and UF and FENa on the other hand. However, better correlations were observed between UNT-pro-BNP, URLX, UUro, and UF and between NT-pro-BNP, RLX, and FENa.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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An increasing number of studies point to a role of RLX in cardiac failure (5, 7). Dschietzig and coworkers (5) have shown an increase in plasma levels and myocardial mRNA expression of RLX in relation to the severity of disease in patients with cardiac failure. They showed that the heart is a major source of circulating RLX during this condition and that the plasma concentrations of this peptide are related to cardiac filling pressures (5), suggesting that the plasma levels of RLX are related to the pathophysiological events of congestive heart failure (CHF): myocardial distension and fluid overload. We thus hypothesized that the secretion of RLX may also be increased by myocardial stretch and may hence, comparable to ANP (10, 20), also react to variations in posture or acute volume loading in healthy individuals.
However, based on the findings of the present study, we have to reject this hypothesis. During the tilting protocol, plasma RLX levels were not affected by posture changes. Hormone levels were significantly lower at 1500 compared with baseline values and increased rapidly back to baseline levels from 1500 to 1600 in both groups. With respect to the highly comparable course in both groups, this may not be explained by tilting per se. Additionally, with respect to the unchanged levels of RLX in the control group of the volume-loading protocol, this may not be explained by circadian rythmicity and hence warrants further study. Additionally, no changes in plasma RLX levels were detectable during volume loading, despite the long observation period. Taken together, these findings more or less rule out that acute variations in TBV are associated with changes in plasma RLX levels in healthy, male subjects.
The plasma RLX levels observed in our study were tremendously higher (30–50 pg/ml) than the normal values (2.5 pg/ml) and the values for severe CHF patients (17 pg/ml) in the study mentioned above (5). Of note, analyses of RLX levels in our study were determined with the same immunoassay and in the same laboratory and were highly reproducible in comparison between the control and the volume-loading group. Because none of the subjects in our study had overt heart failure, according to physical examination, and low NT-pro-BNP levels also ruled out even discrete myocardial disease, on a first glance, these findings questions the data presented by Dschietzig et al. (5). However, in contrast to these investigators, we used aprotinine for inhibition of peptide degradation during blood sampling, a fact that may explain the higher plasma RLX levels in our study.
The course of NT-pro-ANP during tilting and the accompanying changes in LAD show that the changes in position were sufficient to induce relevant changes in these parameters and hence in TBV, at least during the second tilting period. However, no tilting-induced changes in other hemodynamic parameters or in NT-pro-BNP were detectable.
It is long known that dietary sodium loading increases the plasma levels of ANP (11) and BNP (13). This may account for the discrete and short-lasting increase in NT-pro-ANP levels in the control group during the fluid-loading protocol. Comparably, the moderate increase in NT-pro-BNP levels during the tilting protocol in both groups and the increase in NT-pro-BNP levels throughout the fluid-loading protocol in the control group are supposed to be an effect of the standardized breakfast given at 915.
The course of NT-pro-ANP during volume loading is in line with the well-known effects of an acute intravenous saline load on atrial secretion of ANP (20). Only one study has investigated the effects of an intravenous sodium loading by saline infusion on BNP levels in humans (12) and did not find any effect on plasma levels of BNP. Because the observation period in this study was only 1 h, any later increase in BNP levels would have been missed. Consequently, the delayed increase in NT-pro-BNP in our study is an important new finding. Besides the general implications for our understanding of the natriuretic peptide system, this may be relevant for the interpretation of NT-pro-BNP levels as a hormonal marker for the severity of myocardial failure at the bedside, because sodium-containing fluids are frequently used in the clinical setting.
As expected, volume loading induced an immediate rise in UF and FENa without accompanying changes in glomerular filtration rate. Interestingly, these effects were accompanied by significant increases in the URLX and UNT-pro-BNP, whereas only a marginal increase in UUro was observed. Of note, the hormonal excretion of hormones was adjusted for UF by expressing the data relative to creatinine excretion. Hence the excretion pattern cannot be explained simply as a washout in face of an increase in tubular flow and UF. Moreover, the different course of plasma NT-pro-BNP and the unchanged plasma RLX levels suggest that the peptides detected in urine were kidney derived.
RLX as well as BNP have previously been reported to be detectable in urine (19, 23); however, in contrast to Uro, which has been shown to be produced only in the kidney and which has been suggested to play a major role in human sodium homeostasis (8), information on the physiological role of urinary RLX and BNP is sparse. Urinary RLX has been shown to be modified by estrogen therapy in women and has thus been used to quantify the induction of urinary vasoactive markers during contraceptive use (19). Urinary BNP has been shown to be increased in patients with renal (23) and heart failure (17). No data on a possible coupling between renal function and URLX and urinary excretion of BNP are available.
RLX has been shown to increase glomerular filtration rate and sodium excretion in conscious rats, even in the nonpregnant state (2–4). However, in contrast to the well-described distribution of natriuretic peptide receptors in the kidney (14), the distribution of RLX receptors in the kidney is unknown. RLX has been convincingly shown to induce renal vasodilatation and hence a decrease in renal vascular resistance (2, 4). However, it is not known whether additional factors, i.e., tubular effects leading to an inhibition of sodium reabsorption, or other humoral systems are involved.
The different time course of plasma and urinary NT-pro-BNP levels during volume loading more or less excludes that the NT-pro-BNP levels detectable in urine are of plasmatic origin. Comparably, the lack of correlation between UNT-pro-BNP and UF and the minor relationship between UNT-pro-BNP and FENa contradict that the immediate renal response to volume loading may be attributable to variations in plasma NT-pro-BNP levels. Consequently, it is more plausible that the increase in sodium excretion is either related to changes in central hemodynamics (LAD) or ANP and/or mediated by the urinary derivatives NT-pro-BNP or RLX.
In conclusion, the data of the present study show that acute variations in body position and acute intravenous volume loading do not alter plasma levels of RLX in healthy male volunteers. This contrasts with pronounced effects of these maneuvers on the plasma levels of the NT-pro-ANP and NT-pro-BNP and suggests that, despite being increased in relation to filling pressures in CHF, RLX does not react like an acute filling pressure-dependent hormone in healthy humans. However, the association between URLX and urinary excretion of sodium suggests a role of kidney-derived RLX in human sodium and fluid homeostasis that warrants further study.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, Subjects positioned into the feet-down position first and in the head-down position afterward; 
, subjects subjected to an opposite tilting sequence. sup, Supine. Data are given as percentage of baseline (BL) values set to 100% as means ± SE. *Between-group difference, P < 0.05 (ANOVA repeated measures). 
Significant (P < 0.05) difference compared with baseline values (paired Student's t-test).
Significant (P < 0.05) difference compared with values at 1000 (before sodium infusion) (Wilcoxon's matched-pairs test).
