1 Laboratory of Cardiovascular Endocrinology, Consiglio Nazionale delle Ricerche Institute of Clinical Physiology, 56100 Pisa, Italy; and 2 Institute of Biophysics, First Medical Faculty, Charles University, Prague, Czech Republic
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In an attempt to identify and quantify the sites
of atrial natriuretic peptide (ANP) degradation, a new tracer
experiment has been developed.
125I-ANP was injected as a bolus
just upstream from the right atrium, and blood was sampled from two
different sites (pulmonary artery and aorta) in eight cardiac
patients. Data were analyzed using a physiologically based circulatory
model consisting of three blocks in series (right heart, lungs and left
heart, and periphery) supplied by the same flow (cardiac output,
measured by thermodilution); the extraction coefficients of the three
blocks and of the whole body could be determined from the areas under
tracer concentration curves in plasma (AUCs). The values for AUCs
(means ± SD) were 64.8 ± 9.4 and 65.5 ± 10.7%
dose · l1 · min
1
for pulmonary artery and aorta curves, respectively; the area under the pulmonary artery curve could be subdivided into the area
under the first-pass curve (30.6 ± 4.7% dose · l
1 · min
1)
and the area under the recirculating curve (34.0 ± 7.7%
dose · l
1 · min
1).
The metabolic clearance rate of
125I-ANP, computed as dose divided
by the area under the recirculating curve, was 3.1 ± 0.7 l/min, and
the whole body extraction was 47.6 ± 6.6%. In our patients with
myocardial dysfunction, neither right heart block nor lungs and left
heart block significantly extracted ANP, and periphery block
accounted for almost all removal of the hormone from the blood.
atrial natriuretic factor; tracer method; metabolic clearance rate; heart failure
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATRIAL NATRIURETIC PEPTIDE (ANP) is a hormone produced and secreted into the blood by the heart; it has several biological effects, such as natriuresis, vasorelaxation, hypotension, and neuromodulation (7, 20). This hormone seems to play an important role in the natural history of heart failure, mainly by counteracting the detrimental effects of activation of the vasoconstrictor sodium-retaining system (3, 7, 17, 20).
Extensive studies in animals and humans have documented that ANP is secreted into the circulatory system via the coronary sinus into the right atrium and then rapidly degraded and removed from the blood (7, 20). Although the kidney and lungs have been considered to be major sites of ANP removal, little is known about the contributions of the various organs or tissues to ANP clearance from the blood in vivo in humans.
Studies of ANP kinetics (as well as the kinetics of other rapidly degraded, biologically active molecules) have been carried out by a standard experimental protocol in which labeled or unlabeled hormone is administered (by constant infusion or bolus injection) and the corresponding concentration of the hormone is measured in peripheral venous blood (10-13, 26).
Metabolic clearance rate (MCR), the sole parameter describing the rate of disposal at whole body level, is computed by the compartmental (or the so-called noncompartmental) approach (8); in any case, the measured plasma concentration of the substance from a single sampling site is considered to represent the whole intravascular compartment. A uniform plasma distribution is, however, approximately reached only for the slowly degrading system(s), in which the mixing process, ensured by blood flow, is rapid with respect to the degradation rate or, in other words, when MCR is very small relative to plasma cardiac output. However, in the case of rapidly degraded molecules (such as ANP), significant differences in plasma concentrations are measured upstream and downstream from various organs involved in hormone degradation. Indeed, arteriovenous differences have been reported and used to compute hormone extraction values from several circulatory districts (e.g, liver, kidney, lungs) (1, 9, 15, 16, 19, 21, 22). From this, it can be deduced that, after tracer bolus injection, different areas under concentration curves (AUCs) have to be measured upstream and downstream from circulatory ANP-degrading districts and that these different AUCs can be exploited to quantitate extraction values.
To verify this idea, a new experimental protocol has been developed and carried out in a group of cardiac patients undergoing a complete hemodynamic study to evaluate their cardiac disease. The tracer experiment necessitates a bolus injection of 125I-ANP into the right atrium and sampling of concentration curves in the pulmonary artery and aorta. The use of low-rate continuous (integrated) sampling during the first 130 s of the experiment makes it possible to define accurately the earliest part of the pulmonary arterial concentration curve and, thus, distinguish the first pass of the injected bolus from the recirculating curve. Independent measurement of cardiac output was simultaneously obtained by thermodilution.
Because the classical compartmental approach is not suitable for interpreting the extended set of experimental data, we used a more physiological circulatory model that does not assume a uniform intravascular distribution of the hormone. The new circulatory model for describing ANP disposal consists of three blocks connected in series: 1) right heart block, 2) lungs and left heart block, and 3) periphery block. The model can be defined by the three experimentally determined curves: 1) the first-pass curve sampled in the pulmonary artery, 2) the recirculating curve sampled in the pulmonary artery, and 3) the aorta curve.
We report a detailed description of the model, how it works, and the results from a preliminary application in a group of cardiac patients and compare the results with those of standard analysis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental Subjects and Hemodynamics
Experimental subjects. Eight normotensive cardiac patients were enrolled in the study. All the patients were subjected to a complete baseline cardiological evaluation, including physical and X-ray examination, two-dimensional echocardiography, and radionuclide angiography. The patients underwent the hemodynamic study because of their cardiac disease. Their main clinical parameters are reported in Table 1. Because it was not possible to apply this experimental protocol to normal subjects for ethical reasons, we studied patients with a wide range of myocardial involvement (from mild to severe, i.e., left ventricular ejection fraction ranging from 30 to 15%) (Table 1).
|
Hemodynamics. The study was carried out in the hemodynamic ward of the Consiglio Nazionale delle Ricerche Institute of Clinical Physiology after selective left and right coronary angiograms were performed using the Judkins technique (patients 1-5). No premedication was given, and no heparin was used (catheters were regularly flushed with boluses of normal saline). At least 30 min were allowed between coronary angiography and the beginning of the study to minimize any possible interference of the contrast medium with the metabolic parameter.
In all patients a 7-Fr triple-lumen flow-directed balloon-tipped catheter was inserted transcutaneously through the right femoral vein and advanced to obtain right atrial, pulmonary arterial, and pulmonary capillary wedge pressures. Cardiac index was calculated as the ratio of the mean of at least five thermodilution cardiac output measurements to body surface area (m2). After hemodynamic assessment, the flow-directed catheter was removed, and a 7-Fr multipurpose multiple-side-hole catheter was positioned in the pulmonary artery. A 5-Fr pig-tail multiple-side-hole catheter was advanced via the ascending aorta through the introducer positioned in the right femoral artery for diagnostic study in five patients (patients 1-5) submitted to right and left heart catheterization. A three-way stopcock was used to connect each catheter to the pressure transducer and to draw blood or fill the catheter with saline flush solution. Pulmonary arterial pressure, aortic pressure, and one electrocardiographic lead were continuously monitored. Blood samples were simultaneously drawn from the pulmonary artery and ascending aorta. In patients 6-8 a simplified protocol was used consisting of right catheterization and pulmonary arterial blood sampling only.Preparation of the Tracer
Synthetic humanExperimental Protocols of Tracer Studies
During the hemodynamic study, two blood samples (4-5 ml) were collected from the pulmonary artery and the aorta for measuring baseline plasma concentrations of native ANP. A known amount (~80 µCi, corresponding to 110 ng of 125I-ANP, i.e., ~10-15% perturbation of the initial distribution pool of ANP) (11) of freshly prepared tracer (see above) was then intravenously injected as a bolus via a catheter for isotope injection inserted percutaneously from the antecubital vein to near the junction of the superior vena cava and the right atrium. After injection, blood samples were simultaneously collected from the aorta and the pulmonary artery. To make possible a reliable definition of the area under the concentration curve of the tracer during the first few minutes after injection, "integrated" samples were obtained by a continuous withdrawal of blood using a computerized and programmable automatic collector specifically developed for this purpose by the Electronics Unit of the Consiglio Nazionale delle Ricerche Institute of Clinical Physiology. The device consists of 1) a peristaltic pump, 2) a microcontroller, which operates two drivers for piloting two stepping motors, 3) an electronic apparatus for piloting an electromagnet to change the row of tubes, 4) an operating panel with display to program and visualize the work cycle, and 5) an electronic apparatus with an emergency push button to stop the work cycle if necessary. Thirteen 10-s integrated blood samples (1.2 ml each) were simultaneously collected from the aorta and pulmonary artery throughout the first 130 s; the remaining part of the curve (from 130 s to 30 min) was described by at least five "discrete" blood samples of 5 ml, typically taken at 3.5, 8, 15, 20, and 30 min. The larger volume of the five discrete samples was necessary to allow a reliable measure of ANP concentration also through the final part of the curve, where ANP activity is extremely low.A volume of 0.9% NaCl solution equal to the volume of blood withdrawn was infused. The blood samples were immediately put into ice-chilled disposable polypropylene tubes containing aprotinin (500 kallikrein-inactivating units/ml plasma) and EDTA (1 mg/ml plasma), and the plasma was rapidly separated in a refrigerated centrifuge at 4°C.
Extraction, Purification, and Measurement of Labeled ANP in Plasma Samples Collected During the Kinetic Studies
Extraction and purification of labeled ANP from plasma by HPLC. All plasma samples were loaded onto Bond Elut C18 cartridges (Analytical International, Harbor City, CA) activated with 2 ml of methanol and washed with 4 ml of 1% trifluoroacetic acid (TFA). After a 10-ml washout with 0.1% TFA, labeled peptides were eluted with 3 ml of a solution containing 99:1 methanol-TFA. The collected effluent was evaporated using a vacuum centrifuge, and the samples were successively reconstituted and subjected to HPLC, as previously described (5, 11). To measure the recovery of labeled ANP throughout the extraction and purification procedures and to take into account possible in vitro labeled ANP degradation in blood, after sample collection, a known amount of purified 131I-ANP (~3,000-4,000 cpm) was added as internal standard to each polypropylene tube before the start of blood collection (5, 11).
Gamma counting. The 125I and 131I activities were measured in a gamma counter (1282 CompuGamma CS, LKB Wallac, Turku, Finland) with an efficiency of 54 and 60%, respectively; the counting time was 20 min for each fraction, and the operating conditions were chosen so as to obtain a high sample-to-background ratio. After background subtraction, the measured 125I counts were corrected for 131I spillover into the 125I channel (which was 6% under the chosen conditions) (5, 11).
Computation of 125I-ANP Blood-Plasma Partition Factor
Because labeled ANP concentrations were measured in plasma, directly measured blood flow (cardiac output) had to be corrected by multiplying it by the ANP blood-to-plasma ratio, as previously reported (13). The partition of a known amount of labeled ANP added to 3 ml of blood (drawn immediately before the tracer injection) between plasma and cells (e.g., erythrocytes, leukocytes, platelets) was measured in each patient after the common procedure of centrifugation and separation utilized for the kinetic study. On average, this factor was 0.65 ± 0.041.ANP Assay
Plasma samples, immediately separated by centrifugation, then frozen and stored in various aliquots at ![]() |
DATA ANALYSIS |
---|
Description of the Kinetic Model
The analysis is based on the circulatory model depicted in Fig. 1. The ANP body system is schematized as three blocks: right heart, lungs + left heart, and periphery. In more detail, the periphery block can be considered as a parallel of various organs and/or circulatory districts. The same flow (F) circulates through the three blocks connected in series; because the concentration of tracer ANP is measured in plasma and not in blood, the conversion factor is indirectly included in F, which is computed by multiplying cardiac output by the blood-to-plasma concentration ratio and is referred to here as plasma cardiac output. The tracer injection point, the sampling points used in the actual protocol (pulmonary artery, aorta), and the sampling point of a standard protocol (peripheral vein) are indicated in the scheme.
|
Under the usual assumptions of linearity and stationarity, each block
is characterized by its unitary impulse response
[f(t); this function, also called transit time-density function (ttdf), is
normalized, i.e., f(t)dt = 1] and by its extraction coefficient (E) or, alternatively, by
its transmission coefficient (T). As usual, E is defined as the amount
of substance degraded within the block and expressed as a fraction of
the total amount that enters the block. T represents the remainder,
which, not being extracted, escapes intact from the block and
recirculates into the body. Because E + T = 1, E or T is used
interchangeably, and one is preferred over the other, because the
actual equation can be written in a simpler or more suggestive way. E
and T are occasionally reported as a percentage, rather than a
fraction.
We recall that when the substance is carried by a constant F and
concentrations are constant in input
(Ci) and output
(Co), then from the definition
it follows that E = (Ci Co)/Ci
and T = Co/Ci.
If input is a bolus (corresponding experimentally to a concentration
curve with a peak), the amount entering the system is
AUCiF (where
AUCi is the AUC in input).
Moreover, the amount leaving is
AUCoF (where
AUCo is the AUC in output), and
therefore E = (AUCi
AUCo)/AUCi
and T = AUCo/AUCi.
Let us define frh(t), Trh and fp(t), Tp and fl + lh(t), Tl + lh, and the ttdf and transmissions of right heart, lungs + left heart, and periphery blocks, respectively. It is convenient to consider the whole body as a single perfused organ with "open-loop" extraction (Ewb) and flow equal to F. The situation can be easily imagined by cutting the circulation at some point, e.g., at the level of the pulmonary artery; Ewb is therefore the fraction of the substance entering the pulmonary artery that is degraded throughout the body system in one cycle (and that does not return to the pulmonary artery). Because the whole body block is made up of three blocks arranged in series, its transmission is the product of individual transmissions, i.e.
![]() |
(1) |
![]() |
(2) |
Description of the Experimental Curves
After bolus injection of the tracer [dose (D)] upstream from the right heart block, concentration curves are measured in the pulmonary artery [cpulm(t)] and aorta [caorta(t)]. The typical shape of the two curves is shown in Figs. 2-4. According to the circulatory model, these curves are thought to be generated by the summation of a series of peaks
![]() |
(3) |
![]() |
(4) |
|
|
|
Because of frequent sampling, it was possible to split the whole concentration curve generated in the pulmonary artery into a first-pass curve [cpulm,1p(t)] and a recirculating curve [cpulm,rc(t) = cpulm,2p(t) + cpulm,3p(t) + ...]
![]() |
(5) |
![]() |
(6) |
Computation of the Extraction Coefficients of the Model
With regard to hormone disposal, the model is defined by three independent transmission coefficients: Trh, Tl + lh, and Tp (plus Twb = Trh Tl + lh Tp).The three transmissions (and hence the 3 extractions) are computed from AUCpulm, AUCpulm,1p, and AUCaorta and plasma cardiac output (F) using the following equations.
Extraction of the right heart. The first pass of tracer in the pulmonary artery [Fcpulm,1p(t)] can be viewed as the output of the right heart block after bolus injection (upstream to right heart block) of the dose D; by definition it is
![]() |
(7) |
![]() |
![]() |
(8) |
![]() |
(9) |
Extraction of the whole body block. The whole curve measured in the pulmonary artery is the sum of the individual passes of the bolus that recirculates (see Eq. 3), and in terms of areas (i.e., integrating from 0 to infinity)
![]() |
![]() |
(10) |
![]() |
(11) |
![]() |
(12) |
![]() |
(13) |
![]() |
![]() |
(14) |
![]() |
(15) |
![]() |
(16) |
Extraction of the lungs + left heart. Let us consider the curves measured in the pulmonary artery and the aorta as the sum of the individual passes (see Eqs. 3 and 4). The first pass in the aorta curve can be written as the output of the lungs + left heart block produced by the first pass in the pulmonary artery as input; i.e.
![]() |
(17) |
![]() |
(18) |
![]() |
(19) |
![]() |
(20) |
Extraction of the periphery block. The transmission (Tp) and, hence, the extraction can be obtained from Eq. 1, once Trh, Twb, and Tl + lh have been computed (from Eqs. 9, 16, and 20).
Alternatively, the transmission coefficient of the periphery + right heart block (TpTrh, defined as the series of periphery and right heart) is computed as the ratio of the area under the recirculating curve in the pulmonary artery (output curve) to the area under the aorta curve (input curve); i.e.
![]() |
(21) |
Extraction coefficients computable by use of a simplified experimental protocol (right heart catheterization only). By this simplified experiment, only the pulmonary curve is available; it can be split into first pass and recirculating curves, from which AUCpulm, AUCpulm,1p, and AUCpulm,rc are computed. AUCaorta is not available.
From the foregoing equations, the following parameters can be computed: 1) transmission (extraction) of the right heart block (Eq. 9) and 2) transmission of the whole body block (Eq. 15). Tl + lh and Tp cannot be further evaluated unless some assumption is made.Relationship Between Ewb and Metabolic Clearance Rate
According to the circulatory model, the overall degradative capability of the metabolic system is quantitated by the extraction coefficient Ewb. Because the whole body is viewed as a perfused organ with flow F, it is reasonable to expect that its metabolic clearance rate is obtained from extraction and flow according to the well-known relationship
![]() |
(22) |
On the other hand, the MCR currently reported in the literature on hormone kinetics is obtained by dividing the bolus administered dose by the AUC sampled in a peripheral vein (AUCv)
![]() |
(23) |
![]() |
(24) |
It is therefore most interesting to derive the relationship between MCR and MCR*; to do this, we also express MCR* in terms of AUCs. We recall two equations already derived: Eq. 8 states that AUCpulm,1p is equal to the dose that reaches the pulmonary artery (DTrh) divided by F, and Eq. 14 states that the whole area AUCpulm is equal to the AUCpulm,1p divided by Ewb. Combining Eqs. 8 and 14, we obtain
![]() |
(25) |
Multiplying the numerator and the denominator of Eq. 25 by Twb and taking into account that AUCpulmTwb = AUCpulm,rc (Eq. 16), we obtain
![]() |
(26) |
When Eq. 26 is compared with Eq. 24, it follows that
![]() |
(27) |
Generalized Relationship Between MCR* and AUC
MCR* can also be calculated from AUCv (schematically represented in Fig. 1 as the effluent of the parallel subblock P1 perfused by flow F1). At variance with central sampling sites (pulmonary artery and aorta), the peripheral sampling site is perfused by a fraction of the total plasma cardiac output. An equation analogous to Eq. 8 can be written for the area under first pass in the peripheral vein
![]() |
(28) |
![]() |
(29) |
![]() |
![]() |
(30) |
![]() |
(31) |
In conclusion, Eqs. 25, 26, and 31 are particular cases of a general relationship
![]() |
(32) |
Simplified Relationships When Right Heart Block Does Not Extract
It is useful to consider the case in which the tracer is not extracted by right heart block; this case seems of practical relevance for the present studies, in which extraction by right heart block was virtually absent.When Trh = 1, Eq. 11 becomes
![]() |
(33) |
Also Eq. 27 is simplified as follows
![]() |
(34) |
![]() |
(35) |
![]() |
(36) |
Estimation of AUCs
Accurate determination of AUCs (of the pulmonary artery and aorta curves) during the earliest minutes of the study (including the 1st-pass peak) relies on continuous sampling technique. The AUC was computed by summing all concentrations of consecutive integrated samples (which are the average concentrations in each sampling interval) and multiplying by the length of the time interval. The area under the remaining portion (normal sampling) of the curve was analytically calculated from a function sum of two exponentials fitted on the experimental points (6-7 points), as previously described (11).Because of frequent sampling in the first minutes, the onset of recirculation in the pulmonary artery could be detected as a change in the descending slope of the initial peak evident when data are plotted on a logarithmic scale. The area relative to the first pass (AUCpulm,1p) was computed as previously described until the onset of recirculation; the area of the tail computed by monoexponential extrapolation of the descending branch of the peak was added.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Native ANP Plasma Levels
The ANP plasma concentrations determined in the pulmonary artery and aorta in all the patients are reported in Table 1. As expected, the highest values were observed in patients with more severe disease, as indicated by their lower ejection fraction and cardiac index associated with higher wedge pressure and New York Heart Association class (patients 1, 4, and 8). If one takes into account the ratio of pairs of samples simultaneously drawn from the aorta and the pulmonary artery for each patient, extraction values range from 39 toTracer Purity
A drop of the injected tracer remaining in the syringe after the injection was collected and tested for purity by HPLC; in all kinetic studies, impurity was <1%. As an example, a typical chromatogram of the dose injected (patient 3) is shown in Fig. 5; only one peak of radioactivity was found, corresponding to the elution pattern of monoradioiodinated ANP (125I-ANP injected and 131I-ANP added just before HPLC to monitor the possible in vitro degradation).
|
Tracer Studies
The time courses of 125I-ANP concentrations measured in the pulmonary artery and aorta from a typical study are reported in Figs. 2-4. The sampling protocol made it possible to obtain a good definition of the radioactivity time course at these two different sites of the circulation. Total AUCs could, therefore, be calculated accurately for both sites. Moreover, the curve sampled in the pulmonary artery could be split into first-pass and recirculating curves (as indicated in Fig. 4), and total area (AUCpulm) could be divided into the respective components AUCpulm,1p and AUCpulm,rc (Table 2).
|
In Fig. 3, 125I-ANP activity sampled in the pulmonary artery is compared with total radioactivity in the corresponding samples. Total radioactivity and 125I-ANP activity during the first pass are practically superimposable (the ratio of 125I-ANP activity to total radioactivity is 99.5 ± 4.3%). A significant amount of degradative products from injected 125I-ANP was found in plasma only after recycling of the labeled dose (Fig. 5B). If we start at the onset of recycling [at 2 min 125I-ANP activity is 77 ± 7% (SD) of total radioactivity, n = 8], the difference between the two curves markedly increases (at 4.5 min 125I-ANP activity is 38 ± 4% of total radioactivity) because of the progressive accumulation of final metabolites (125I-tyrosine, 125I, and/or other radiolabeled metabolites of 125I-ANP) with slower kinetics (5, 11). At the end of the experiment (30 min after tracer injection) 125I-ANP activity was only 5.0 ± 2.1% of total radioactivity.
Total areas under the two curves (calculated from zero to infinity) sampled in the pulmonary artery and the aorta are reported in Table 2 for each study; the area under the first-pass curve and the area under the recirculating curve in the pulmonary artery are also reported. These values, together with plasma cardiac output (Table 3), allow extraction (or transmission) coefficients of the three blocks (right heart, lungs + left heart, and periphery) and of the whole body system (whole body block) to be computed and so to completely define the circulatory model (Table 3).
|
The product of AUCpulm,1p (on average 30.6% dose/l) and plasma cardiac output [on average 3.31 l/min, determined as cardiac output (obtained by thermodilution) multiplied by the measured blood-plasma partition factor, see MATERIALS AND METHODS] allows the amount of ANP that passes through the pulmonary artery and enters the lungs to be computed (on average 99.2% of the injected dose as calculated by weighing the syringe). This value corresponds to the percentage of the dose that is recovered from the pulmonary artery and allows a direct measurement of the ANP transmission coefficient through the right heart block (see Trh in Table 3). The computed mean value of 99.2% indicates that no measurable extraction of ANP took place during the transit through the right heart chambers.
The area under the first-pass peak measured in the pulmonary artery (AUCpulm,1p) was about one-half of the total AUCpulm in all patients (Table 2); as a consequence, Ewb, calculated as the ratio of first pass to total area, was 47.6% on average, with a relatively narrow range (39.9-60.0%).
The mean total curves (from the first 5 studies in Table 2) in the
pulmonary artery and aorta are depicted in Fig. 4; the aorta curve is
delayed relative to the pulmonary curve, and it can be appreciated at a
glance that the two AUCs (AUCpulm
and AUCaorta) are similar. This
impression is confirmed by data in Table 2 (on average 64.8 vs. 65.5%
dose · l1 · min
1
for AUCpulm vs.
AUCaorta), and so the
transmission of the lungs + left heart block was ~100%. Inasmuch as
no measurable extraction was found for the lungs + left heart or the
right heart block, whole body extraction could be entirely attributed
to the periphery block.
The last three studies (patients 6-8 in Tables 1-3) were carried out with a simplified experimental protocol that required right heart catheterization only; by this approach, extraction values of whole body and right heart block can be calculated, whereas no information on lungs + left heart block extraction can be obtained. With regard to whole body extraction and right heart transmission, the results we obtained (52.2 ± 8.8 and 99.7 ± 1.4%, respectively) were superimposable on those of the previous five studies (44.9 ± 3.7 and 98.9 ± 1.2%, respectively). The periphery block can then be considered responsible for the total hormone extraction; therefore, we conclude that, on average, extraction of the periphery block is 47.6% (i.e., equal to average whole body extraction observed in all 8 studies).
MCR Values
Two different values for clearance rate (MCR and MCR*) are reported in Table 3 for each case; the first (MCR) was calculated as D/AUCpulm,rc and is directly comparable (see DATA ANALYSIS) to previously reported values (6, 9-13, 26). All these values, reported by many authors and by us (6, 9-13, 26), were computed as the ratio of the entire dose to AUCv; this area is very similar to AUCpulm,rc, i.e., the area under the pulmonary curve after subtraction of the first-pass curve. In accordance with these considerations, the mean values of MCR (3.06 l/min) were similar to those previously reported (10, 11).On the other hand, MCR* is the clearance rate defined as the perfused body extraction (Ewb) multiplied by the plasma cardiac output (F). The values of MCR* reported in Table 3 have been computed as follows: MCR* = D/AUCpulm, which is theoretically correct (see DATA ANALYSIS) when 100% of the injected dose reaches the pulmonary artery. In each case, the MCR* was similar to Ewb times F; on average, MCR* was 1.57 l/min, which coincides with the product of average values of F and Ewb (i.e., 3.31 × 47.6% = 1.57 l/min).
The MCR* was about one-half of MCR (on average 1.57 vs. 3.06 l/min, with MCR/MCR* = 1.57/3.06 = 51.3%). Indeed, from theory (see DATA ANALYSIS) it can be derived that MCR*/MCR is equal to the perfused body transmission coefficient (Twb), which was 52.4% on average.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Why Resort to the Circulatory Model, and How Does the Circulatory Model Work?
A very popular experimental protocol for describing the kinetics (renewal) of endogenously produced substances implies the bolus injection of tracer (generally in a peripheral vein) and the plasma sampling of only one disappearance concentration curve (generally from a different peripheral vein or more rarely from an artery district). On the basis of the compartmental model (or of the so-called noncompartmental approach) (8), a single parameter (MCR) is computed to quantitate the degradation rate at the whole body level; whatever the approach, MCR is calculated as the ratio of the injected dose to the area under the single disappearance curve (MCR = D/AUCv).This approach, which has the advantage of being very practicable, has been applied, essentially unmodified, to the study of numerous endogenously produced substances, despite their very different kinetic characteristics (8). Measured MCR values show extreme variations. For example, an MCR of 0.7 ml/min has been reported for thyroxine (T4) (18), whereas the MCR of several produced hormones (e.g., all gonadal and adrenal sexual hormones and aldosterone) is 1,000-1,500 ml/min (i.e., on the same order as plasma cardiac output). Furthermore, MCR values as large as 3,000 ml/min or more have been found for ANP (7, 11, 20). It is, however, intuitive that the wide differences in metabolic behavior (e.g., slowly degraded T4 vs. rapidly degraded ANP) imply that experimental data produced by the bolus injection protocol should be interpreted in somewhat different ways.
Indeed, when labeled hormone is bolus injected (or alternatively when native hormone is secreted in a burst), it can be predicted that the AUC can be the same along the whole intravascular compartment (at least within the limits of precision of the measurement) when the degradation processes are relatively slow with respect to the mixing ensured by circulation (e.g., for T4). However, for rapidly degrading systems (such as ANP) the AUCs, sampled at different circulatory districts, will be different. The different AUCs measured at different sampling sites in rapidly degrading systems, as opposed to the slowly degrading system in which only one AUC is measured, pose some problems in interpretation. In fact, one would be puzzled by the different MCR values produced by the simple formula D/AUC (8) when different values of AUC are introduced.
The experimental protocol of the present studies allows an extended set
of data to be collected after the bolus injection of tracer ANP, i.e.,
AUCs measured at different sampling sites together with direct
measurement of cardiac output. As expected for a substance with a very
high clearance rate, large differences in the AUCs were observed in all
studies (Table 2); in particular, AUCaorta (65.5%
dose · l1 · min
1),
i.e., upstream from the periphery, was about twice that of AUCpulm,rc (34%
dose · l
1 · min
1)
measured downstream from the periphery.
Although these new experimental data give a more detailed description of the ANP system, they could not be analyzed using the compartmental model (or the so-called noncompartmental approach) used in previous works on ANP kinetics (6, 7, 26) and also adopted in our previous studies (10-13). These models of whole body ANP kinetics are extremely simplified and assume (more or less explicitly) that the intravascular space behaves as a single initial distribution compartment; therefore, they cannot account for different disappearance curves of tracer sampled in different sites of the intravascular space. Moreover, hemodynamic factors cannot be explicitly addressed by these approaches. For these reasons, we resort to the circulatory model previously developed and mostly used in different applications for exogenous substances (2, 14, 24, 25).
With respect to the compartmental approach, the circulatory model gives a more physiologically based description of the kinetics of rapidly degraded substances (such as ANP) directly secreted into the bloodstream. The parameters that describe the degradation of ANP are the extraction (or transmission) coefficients associated with the individual blocks and the cardiac output. At the whole body level, a single parameter, Ewb, extraction of open-loop "perfused body," is defined. The analysis is based on the indicator-dilution principles, avoiding any assumption of instantaneous mixing into a central (plasma) compartment, which is clearly unrealistic for rapidly degraded substances. Transmission coefficients for the individual block (or for the whole body) are computed as the ratio of the AUC measured downstream to that measured upstream from the pertinent block (or the perfused body). An obvious advantage is that different AUCs measured in plasma at different sites can be interpreted, and these differences can be exploited to compute extraction coefficients of individual districts, provided that pertinent experimental data (upstream and downstream curves) are available.
An additional advantage of the circulatory model is that the effects of hemodynamics on the metabolism of the substance can be made evident. Although in a compartmental model the degradation is described by the single parameter MCR, in the circulatory model the degradation is associated with two parameters, Ewb and F, linked by the well-known relationship: MCR* = EwbF. By this definition, MCR* depends on two factors: extraction, which represents an intrinsic characteristic of the body, and plasma cardiac output, which represents the contribution of hemodynamic factors. In fact, for hormones with very fast kinetics (such as ANP), changes in cardiac output can affect the rate of removal (flow-limited removal), a behavior that cannot be accounted for by compartmental analysis in which circulation is not considered.
More explanation is needed to describe why, in rapidly degrading systems, MCR*, defined as EwbF, differs from the very popular and much referred to MCR = D/AUCv, computed from a peripheral venous curve. In the presence of different plasma AUCs measured in different circulatory districts, the simple formula MCR = D/AUC cannot be used without more explanation, since confusingly different values of MCR are generated. It can be shown (see DATA ANALYSIS) that the general relationship to be used is MCR* = DTx/AUCx, in which the dose is corrected for the transmission coefficient Tx from the injection site to the generic (x) sampling site of the curve used to obtain AUCx. Indeed, by introducing different AUCx into this general formula, the same value of MCR* is calculated, since the corresponding transmission coefficient Tx takes into account the fraction of the dose extracted during the passage from the administration site to the sampling site.
As an example from the present experiment, the finding that MCR was about twice MCR* is a consequence of MCR being computed as the ratio of the entire dose to the AUC measured in the periphery. According to the circulatory model, this formula is not correct in the case of a rapidly degraded hormone, such as ANP; in this case, it should be taken into account that only 52.4% of the administered dose reaches the periphery because of rapid extraction in a single pass.
The relationship linking MCR* with MCR can also be written as follows:
Ewb = MCR/(MCR + F) (see
DATA ANALYSIS); in this form the
relationship allows Ewb to be
derived from MCR when an estimate of F is available. It is easily seen
that when we measure an MCR value (3.06 l/min on average) similar to
plasma cardiac output F (3.31 l/min on average), as in the case of ANP,
the Ewb approximates 50%. The
above relationship is correct for rapidly and slowly renewed systems;
however, when MCR F (i.e., in slowly renewed systems such as that
of the thyroid hormone T4) (18),
the relationship is approximated by the simpler equation
Ewb = MCR/F, so that MCR and MCR*
practically coincide. It is also important to emphasize that the
complete relationship always gives a correct value of extraction, even
when MCR is larger than F; however, in this case, the simplified
relationship Ewb = MCR/F would
produce a puzzling value of extraction >1.
Circulatory Model and Constant-Infusion Protocol
The use of the circulatory model can easily be extended to the protocol in which tracer is administered by constant infusion (13). The main difference is that, after a proper equilibration period, different steady-state tracer concentrations (instead of different AUCs, as in the case of bolus administration) are measured upstream and downstream from various extracting circulatory districts. The equations are very similar to those derived here, where the corresponding steady-state levels substitute for AUCs. In particular, transmission coefficients are computed as the ratio of tracer levels measured downstream to those measured upstream from the district. A relationship was derived previously (13) to convert from peripherally measured MCR [computed as the ratio of infusion rate (IR) to steady-state concentration in peripheral vein (Cv), i.e., MCR = IR/Cv] to Ewb, if a value for F is available. We previously reported values for MCR and Ewb of labeled ANP determined by means of a relatively simplified constant-infusion protocol (13); these values are similar to those reported in the present study by using the bolus injection protocol and sampling in the pulmonary artery.Indeed, one important limitation of the approach presented previously (13) is that it was based on a direct measurement of only Cv (where steady-state concentration in peripheral vein was considered to be the output of the perfused body). The concentration of tracer ANP in pulmonary artery, input of the perfused body, has been reconstructed as peripheral venous concentration added to the ratio of infusion rate (experimentally controlled) to measured plasma cardiac output (i.e., Cv + IR/F) under the assumption of negligible right heart extraction (13). It should be emphasized that by using the bolus protocol the input and the output from the perfused body are accurately and directly measured by the curve sampled in the pulmonary artery and separated as first-pass and recirculating curves. This is not possible, even if pulmonary samples were available, for infusion studies, since output steady-state concentration of perfused body (steady-state concentration at the pulmonary artery level) cannot be experimentally resolved from input function.
A major advantage of the experimental protocol in which tracer is infused at a constant controlled rate is that it simulates the endogenous condition in which the native substance is secreted at a constant rate. It seems more useful to continuously monitor the degradation rate and extraction during stimulatory tests that produce, for instance, variation in the endogenous secretion of the hormone (13).
Accuracy in the Computation of AUCv
In rapidly degrading systems the accurate estimate of AUCv from the concentration curve measured in a peripheral vein after bolus injection is not a simple task and requires the adoption of a suitable sampling protocol. Generally speaking, the higher the degradation rate, the more frequent should be the sampling of the earliest part of the concentration curve. In fact, when MCR is of the same order as plasma cardiac output, ~50% of the tracer is extracted in every single pass through the whole body (which requires a mean recycling time of ~1 min). It is therefore intuitive that if the first sample is taken starting from 3-5 min after injection, a considerable amount of tracer is already degraded, and therefore an accurate estimate of AUC is unlikely to be reconstructed regardless of the mathematical approach.From our "recirculating" curves of ANP, we observed that a large fraction (40-50%) of the area is relative to the first 5 min (Fig. 5), and so an insufficient sampling in this early period could produce large errors in AUCv. On the other hand, the area extrapolated from 30 min to infinity represents only a minor contribution to the total area (<5% of total area on average in the present studies), and therefore the sampling protocol is less critical.
According to these considerations, it is advisable when quantifying ANP (or similar substances) to obtain at least four or five samples in the first 5 min, thus avoiding the integral of the sharp peak of the initial part of the curve being inaccurately computed due to insufficient sampling. In any case, it is not advisable to use exponentials with nonzero intercepts to fit the experimental points as is currently done in compartmental analysis; the use of a function with nonzero intercept derives from the assumption of instantaneous mixing of the tracer dose before an appreciable degradation takes place, and this assumption is untenable for rapidly degraded hormones. Clearly, the backextrapolation to zero of the early experimental points, in contrast to any experimental evidence reported here, induces overestimation of AUCv, in our experience a 20% overestimation of multiexponential fit in respect to trapezoidal integration. In addition, the overestimation can be particularly large when cardiac output is slow and the peak of recirculation is delayed (up to 40% overestimation in our studies).
A better strategy would be to use an integrated sampling (as reported here for the curves sampled in pulmonary artery and aorta), which ensures against loss of accuracy due to insufficient sampling and, at the same time, does not require any interpolating procedure.
Pathophysiological Implications of Kinetic Results
The results obtained with the newly developed kinetic approach applied in the present study provide new information on ANP degradation in humans and, at the same time, could help clarify previous, often contradictory, data. Our data confirm that whole body extraction of the hormone is very high (~50%), as previously reported by others (6, 7, 20) or by us (13) using a different experimental approach (infusion instead of bolus injection). An important finding is that in our patients no measurable extraction of 125I-ANP was found for lungs and heart, so all the hormone-extracting processes take place downstream from the aorta district. Our results seem to be in contrast with previous reports on the role of the lungs in ANP extraction in normal and heart failure states (9, 22, 23). These data, however, need further confirmation in a larger sample of patients with a study design specific for this purpose. A major limitation of this approach is that it is feasible only in patients undergoing a complete hemodynamic study because of their cardiac disease, including a direct measurement of cardiac output (by thermodilution).Finally, an interesting finding is that whole body extraction of ANP could be a relatively stable parameter (47.6 ± 6.6%), even in the presence of large fluctuations of plasma flow (2.7-4.3 l/min) and MCR (2-4 l/min), thus confirming the intrinsic, organ-specific nature of this parameter independent of changes in hemodynamic factors. However, to improve our knowledge of the action and the pathophysiological role of ANP, future studies using this model should be planned to identify sites and mechanisms of ANP clearance from the circulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Stefano Turchi and Franco Cazzuola for technical assistance, Marisa Corfini for dietetic assistance, and Roberta Bertolini for secretarial assistance. In addition, we thank Alessandro Riva and Marco Paterni for setting up the automatic sampling device.
![]() |
FOOTNOTES |
---|
Address for reprint requests: A. Pilo, C. N. R. Institute of Clinical Physiology, Via Savi 8, 56100 Pisa, Italy.
Received 5 March 1997; accepted in final form 13 November 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akaike, M.,
F. Ishikura,
S. Nagata,
K. Kimura,
and
K. Miyatake.
Direct secretion from left atrium and pulmonary extraction of human atrial natriuretic peptide.
Am. Heart J.
123:
984-989,
1992[Medline].
2.
Bischoff, K.
Physiological pharmacokinetics.
Bull. Math. Biol.
48:
309-322,
1986[Medline].
3.
Brandt, R. R., R. Scott Wright, M. M. Redfield, and J. C. Burnett. Atrial natriuretic peptide
in heart failure. J. Am. Coll.
Cardiol. 22, Suppl. A:
86A-92A, 1993.
4.
Clerico, A.,
G. Iervasi,
M. G. Del Chicca,
S. Maffei,
S. Berti,
L. Sabatino,
S. Turchi,
F. Cazzuola,
C. Manfredi,
and
A. Biagini.
Analytical performance and clinical usefulness of a commercially available IRMA kit for the measurement of atrial natriuretic peptide in patients with heart failure.
Clin. Chem.
42:
1627-1633,
1996
5.
Clerico, A.,
G. Iervasi,
C. Manfredi,
S. Salvadori,
M. Marastoni,
S. Turchi,
M. G. Del Chicca,
D. Giannessi,
S. Del Ry,
M. G. Andreassi,
A. Biagini,
and
L. Donato.
Preparation of mono-radio-iodinated tracers for studying the in vivo metabolism of atrial natriuretic peptide in humans.
Eur. J. Nucl. Med.
22:
997-1004,
1995[Medline].
6.
Crozier, I. G., M. G. Nicholls, H. Ikram,
E. A. Espiner, T. G. Yandle, and S. Jans.
Atrial natriuretic peptide in humans: production and clearance by
various tissue. Hypertension 8, Suppl. 2: 11-15, 1986.
7.
Espiner, E. A.
Hormones of the cardiovascular system.
In: Endocrinology (3rd ed.), edited by L. J. DeGroot. Philadelphia, PA: Saunders, 1995, p. 2895-2916.
8.
Gurpide, E.
Tracer Methods in Hormone Research. Berlin: Springer-Verlag, 1975.
9.
Hollister, A. S.,
R. J. Rodeheffer,
F. J. White,
J. R. Potts,
T. Imada,
and
T. Inagami.
Clearance of atrial natriuretic factor by lung, liver, and kidney in human subjects and the dog.
J. Clin. Invest.
83:
623-628,
1989[Medline].
10.
Iervasi, G.,
A. Clerico,
S. Berti,
A. Pilo,
A. Biagini,
R. Bianchi,
and
L. Donato.
Altered tissue degradation and distribution of atrial natriuretic peptide in patients with idiopathic dilated cardiomyopathy and its relationship with clinical severity of the disease and sodium handling.
Circulation
91:
2018-2027,
1995
11.
Iervasi, G.,
A. Clerico,
S. Berti,
A. Pilo,
F. Vitek,
A. Biagini,
M. T. Baratto,
R. Bianchi,
and
L. Donato.
ANP kinetics in normal men: in vivo measurement by a tracer method and correlation with sodium intake.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F480-F489,
1993
12.
Iervasi, G.,
A. Clerico,
A. Pilo,
S. Berti,
F. Vitek,
A. Biagini,
R. Bianchi,
and
L. Donato.
Kinetic study of atrial natriuretic peptide in patients with idiopathic dilated cardiomyopathy: evidence for resistance to biologic effects of the hormone even in patients with mild myocardial involvement.
J. Cardiovasc. Pharmacol.
24:
626-637,
1994[Medline].
13.
Iervasi, G.,
A. Clerico,
A. Pilo,
F. Vitek,
S. Berti,
C. Palmieri,
M. Ravani,
L. Sabatino,
C. Manfredi,
M. G. Del Chicca,
A. Biagini,
and
L. Donato.
Evidence that ANP tissue extraction is not changed by large increases of its plasma levels induced by pacing in humans.
J. Clin. Endocrinol. Metab.
82:
884-888,
1997
14.
Mari, A.
Circulatory models of intact-body kinetics and their relationship with compartmental and non-compartmental analysis.
J. Theor. Biol.
160:
509-531,
1993[Medline].
15.
Northridge, D. B.,
M. P. Jamieson,
A. G. Jardine,
K. J. D. MacArthur,
N. MacFarlane,
and
H. J. Dargie.
Pulmonary extraction and left atrial secretion of atrial natriuretic factor during cardiopulmonary bypass surgery.
Am. Heart J.
123:
698-703,
1992[Medline].
16.
Obata, K.,
H. Yasue,
K. Okumura,
K. Matsuyama,
H. Ogawa,
M. Kurose,
Y. Saito,
K. Nakao,
H. Imura,
and
M. Nobuyoshi.
Atrial natriuretic polypeptide is removed by the lungs and released into the left atrium, as well as the right atrium, in humans.
J. Am. Coll. Cardiol.
15:
1537-1543,
1990[Medline].
17.
Packer, M.
The neurohormonal hypothesis: a theory to explain the mechanisms of disease progression in heart failure.
J. Am. Coll. Cardiol.
20:
248-254,
1992[Medline].
18.
Pilo, A.,
G. Iervasi,
F. Vitek,
M. Ferdeghini,
F. Cazzuola,
and
R. Bianchi.
Thyroidal and peripheral production of 3,5,3'-triiodothyronine in humans by multicompartmental analysis.
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E715-E726,
1990
19.
Rodeheffer, R. J.,
I. Tanaka,
T. Imada,
A. S. Hollister,
D. Robertson,
and
T. Inagami.
Atrial pressure and secretion of atrial natriuretic factor into the human central circulation.
J. Am. Coll. Cardiol.
8:
18-26,
1986[Medline].
20.
Ruskoaho, H.
Atrial natriuretic peptide: synthesis, release, and metabolism.
Pharmacol. Rev.
44:
479-602,
1992[Medline].
21.
Schutten, H. J.,
J. H. Henriken,
and
J. Warberg.
Organ extraction of atrial natriuretic peptide (ANP) in man. Significance of sampling site.
Clin. Physiol.
7:
125-132,
1987[Medline].
22.
Sugawara, A.,
K. Nakao,
N. Morij,
M. Sakamoto,
M. Suda,
M. Shimokura,
Y. Kiso,
M. Kihara,
Y. Yamori,
K. Nishimura,
J. Soneda,
B. Toshihito,
and
H. Imura.
-Human atrial natriuretic polypeptide is released from the heart and circulates in the body.
Biochem. Biophys. Res. Commun.
129:
439-446,
1985[Medline].
23.
Turrin, M.,
and
C. N. Gillis.
Removal of atrial peptide by perfused rabbit lungs in situ.
Biochem. Biophys. Res. Commun.
140:
868-873,
1986[Medline].
24.
Van Rossum, J. M.,
J. E. G. M. de Bie,
G. Van Lingen,
and
H. W. A. Teeuwen.
Pharmacokinetics from a dynamical system point of view.
J. Pharmacokinet. Biopharm.
17:
365-392,
1989[Medline].
25.
Yamaoka, K.,
T. Nakagawa,
and
H. Tanaka.
Recirculatory moment analysis of drugs in man: estimation of extraction ratio and mean cycle time for single systemic and pulmonary circulation.
Chem. Pharm. Bull. (Tokyo)
33:
784-794,
1985[Medline].
26.
Yandle, T. G.,
A. M. Richards,
M. G. Nicholls,
R. Cuneo,
E. A. Espiner,
and
J. H. Livesey.
Metabolic clearance rate and plasma half-life of -human natriuretic peptide in man.
Life Sci.
38:
1827-1833,
1986[Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |