© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Calculation of plasma concentrations of intra-arterially infused compounds in forearm plethysmography
aDepartment of Nephrology, University Hospital Leiden, Rijnsburgerweg 10, 2333 AA Leiden, Netherlands
bDepartment of Pharmacotherapy, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, Netherlands
* Corresponding author. Tel. +31-70-3592000; Fax +31-71-5248118; E-mail: bruning@rullf2.leidenuniv.nl
Received 27 February 1997; accepted 6 August 1997
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Forearm blood flow plethysmography is a widely accepted in vivo technique for pharmacologic and functional studies in peripheral resistance vessels and veins. Pharmacological effects on forearm blood flow (FBF) are usually expressed by means of dose-response relationships. This approach does not consider the influence of variations in FBF on the actual plasma concentrations of compounds infused, and is less suitable for quantitative comparison of the pharmacologic characteristics of different compounds. The aim of this study was to validate an equation to estimate the plasma concentrations of intra-arterially infused compounds. This was done at different levels of FBF, using an indicator dilution technique with constant rate infusions of indocyanine green (ICG) and inulin. Methods: ICG (0.5 mg/min) and inulin (5 mg/min) were infused into the brachial artery in the presence of sodium nitroprusside (10 ng/kg/min; to obtain high FBF), vehicle (0.9% saline; for intermediate FBF), and methoxamine (1 µg/kg/min; for low FBF). FBF was measured using venous occlusion plethysmography in six healthy male volunteers. Plasma concentrations of the indicators, measured in venous blood samples, were compared with the calculated values. Results: Excellent correspondence was observed between calculated and measured plasma concentrations for both ICG and inulin. Venous plasma concentrations of ICG (
95% protein binding) reached steady-state within four min independent of FBF. Alternatively, the time required for venous plasma concentrations of inulin (not bound to protein) to reach steady-state appeared dependent on FBF. Conclusion: Total plasma concentrations of intra-arterially infused drugs can be appropriately estimated at the level of the arterioles by the proposed equation.
KEYWORDS Human forearm; Plethysmography; Plasma concentrations; Indocyanine green; Inulin; Human
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The human forearm is a widely accepted in vivo model for pharmacologic and functional studies in peripheral resistance vessels and veins [1, 2]. Venous occlusion plethysmography, using mercury-in-silastic strain gauges, has proved to be an accurate technique to measure blood flow in the extremities, and has been refined throughout the years [2–5]. The brachial artery is the sole supplying conduit vessel to the forearm. By cannulating the brachial artery in the cubital fossa, just above its bifurcation, the local vascular effects of infused compounds can be studied. To exclude the possibility of systemic effects the noninfused contralateral arm can be used as a control. Concomitantly, blood pressure can be measured through the intra-arterial cannula and heart rate can be derived from an electrocardiogram.
It is customary to express the effects of the infused compounds on forearm blood flow (FBF) in dose-response relationships (see [5]). One shortcoming of this approach is that it does not consider the fact that plasma concentrations of the compounds infused are influenced by variations in blood flow [5]. Furthermore, the use of dose-response relationships is less suitable for quantitative comparison of the pharmacologic characteristics of different compounds. Recently, we have used a equation to estimate the arterial plasma concentrations of intra-arterially administered compounds that enabled us to characterize the muscarinic receptor subtype mediating cholinergic vasodilatation in human forearm resistance vessels, by means of a classical pharmacologic approach [6]. This method has already been used by different authors [5, 7–9].
The aim of the present investigations was to validate the use of such a method for the vascular bed of the human forearm. We performed constant rate infusions with indocyanine green (ICG, Cardio–Green®), having a volume of distribution that is practically equal to the plasma volume due to its high protein-binding capacity [10], and with inulin, which has a volume of distribution that equals that of the interstitial- and plasma-compartments together [11].
In order to assess the influence of different levels of FBF, these compounds were given in three separate experiments, in the presence of a continuous infusion of the vasodilator sodium nitroprusside (SNP, causing a high FBF), saline (causing an intermediate FBF), and the vasoconstrictor methoxamine (causing a low FBF), respectively.
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2 Methods |
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2.1 Subjects
The study was undertaken in six healthy male volunteers (mean age 23 years, range 19–31). Their medical history, physical examination and routine laboratory tests did not reveal any abnormality. The six subjects refrained from smoking, alcohol and caffeine-containing beverages, starting 12 hours before the experiments. The protocol was approved by the Medical Ethics Committee of the Leiden University Hospital and informed consent was obtained from all subjects. The investigation conforms with the principles outlined in the Declaration of Helsinki.
2.2 Procedures
All experiments were performed in a quiet room, kept at 22–24°C. During the experiments the subjects were in the supine position with both forearms stabilized slightly above the level of the heart. After local anaesthesia of the skin (lignocaine 1%) the brachial artery of the nondominant arm was cannulated in the cubital fossa. The cannula (Viggo-Spectramed 4440-4, Swindon, England) was used for the infusion of drugs with a Harvard constant rate infusion pump (Harvard "22", Harvard Apparatus, Ltd., Edenbridge, Kent, England) and for the intra-arterial recording of the blood pressure (i.a. BP) with a Statham P23Id pressure transducer (Gould, Inc., Oxnard, CA, USA). A deep vein in both arms was cannulated (Venflon 1.2 mm, Viggo-Spectramed, Helsingborg, Sweden) for venous blood sampling. Heart rate (HR) was derived from a continuously recorded one-lead electrocardiogram (ECG). During all experiments the forearm blood flow in both arms was measured at 15 second intervals by R-wave triggered venous occlusion plethysmography (Hokanson EC-2 plethysmograph, Hokanson, Inc., Issaquah, WA, USA), using mercury-in-silastic strain gauges and a rapid cuff inflator (Hokanson E-10). A personal computer (Model AT3, IBM, Armonk, NY, USA) extended by an analog-digital convertor (Model DT 2801, Data Translation Inc., Marlborough, MA, USA) was used for R-wave triggered control of the rapid cuff inflator and for on-line analysis of FBF, i.a. BP and HR.
Because the blood flow through the hand is mainly determined by skin arteriovenous anastomoses in the fingers, both hands were excluded from the circulation using small wrist cuffs, inflated to 40 mmHg above systolic blood pressure during all experiments, in order to ensure stable forearm blood flow and arterial plasma concentrations. These cuffs were kept inflated during the entire course of the infusion experiment (i.e. during 21 min). Recordings were started one minute after inflation of these cuffs. Tracings of the ECG, the BP and the FBF were directly recorded on a polygraph (Mingograph 803, Siemens-Elema, Stockholm, Sweden). Forearm and hand volumes were measured by water displacement.
The experiments started at least 45 min after the cannulation of the brachial artery. Between the various infusions the wrist cuffs were deflated and sufficient time was allowed for FBF to return to pre-infusion values. Between all experiments 30–40 minute intervals were applied. To reduce bias due to possible stress caused by occlusion of the hands, all experiments were of equal duration. However, none of the volunteers experienced any discomfort during the experiments.
2.3 Drugs and solutions
The following compounds were infused into the brachial artery: Sodium nitroprusside (E. Merck, Darmstadt, Germany), methoxamine (ICN Pharmaceuticals Holland, Zoetermeer, The Netherlands), indocyanine green (Becton Dickinson, Cockeysville, MD, USA), and inulin (Laevosan-GmbH, Linz, Austria). The compounds were pharmaceutically analysed before use. The drugs were dissolved in 0.9% saline except for SNP, which was dissolved in 5% dextrose. All solutions were aseptically prepared from sterile stock solutions and ampoules on the day of the study and stored at 4°C until used.
2.4 Study protocol
The general design of the study protocol is shown in Fig. 1Fig. 2 and Fig. 3. The recordings of FBF, i.a. BP and HR started three min before the onset of the infusions. The continuous-dose infusions of ICG (0.5 mg/min at 0.2 ml/min), given together with inulin (5 mg/min at 0.2 ml/min), lasted 16 min. The concomitant continuous infusions of either endothelium-independent vasodilator SNP (10 ng/kg/min at 0.4 ml/min), vehicle (0.9% saline at 0.4 ml/min), or the 
1-adrenoceptor agonist methoxamine (1 µg/kg/min at 0.4 ml/min), lasted 20 min and were started 4 min before the combined infusion of ICG and inulin, which was sufficiently long to allow FBF values to reach steady-state. The total amount of the compounds infused during each experiment was 14.8 ml, with a maximal infusion rate of 0.8 ml/min. The experiments were performed in a randomized order.
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; measured 
) and indocyanine green (ICG; calculated 
, measured 
), in the presence of sodium nitroprusside. Steady-state values for forearm blood flow (
) were reached at four min after the onset of infusion of sodium nitroprusside (mean±SEM; n = 6).*=P<0.05.
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Venous blood samples (10 ml each) were taken at five time-points: at –4 min (before the infusion of SNP, vehicle, or methoxamine), at 0 min (before the combined infusion of ICG and inulin), and at 4, 8, and 16 min after the onset of the infusion of ICG and inulin. Plasma samples were taken from the contralateral (noninfused) arm at –4 and at 16 min, to correct for recirculation of ICG and inulin. The total amount of blood taken from each volunteer was approximately 210 ml.
Heparinized blood samples for the determination of inulin were centrifuged for 10 min at 4000 g, and plasma was immediately separated. Plasma inulin was subsequently determined by a fully enzymatic method [12, 13].
The blood samples for the measurement of ICG were allowed to clot, and were subsequently centrifuged for 10 min at 4000 g, after which serum was separated immediately. A dye calibration curve was prepared from each subject's blank serum. To determine ICG serum concentrations absorption was measured at 775 nm (peak absorption; DU-64 Spectrophotometer, Beckman Instruments Inc., Fullerton, CA, USA).
2.4.1 Calculations and statistics
Pre-infusion values were recorded during three min before each experiment. The mean values of FBF, HR, and i.a. BP, from the six consecutive recordings before each sample, were used for analysis. Results are given as mean values±SEM. Wilcoxon's signed rank test for matched pairs was used to evaluate the statistical significance of the data. P-values lower than 0.05 were regarded as significant.
Plasma concentrations (Cplasma in mg/ml) of the infused indicators ICG and inulin were calculated from the rate of drug infusion (IR in mg/min), hematocrit (Hct), forearm volume (V in ml, minus hand volume), and forearm blood flow (FBF in ml/100 ml/min), as follows:
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3 Results |
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Clinical and hemodynamic characteristics of the subjects are given in Table 1. Pre-infusion values of FBF established in the various experiments were all in the same range for both arms. In all experiments no statistically significant changes in HR or i.a. BP were observed.
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3.1 Plasma concentrations at high forearm blood flow
SNP (10 ng/kg/min) significantly raised FBF from 2.55±0.40 to 6.05±0.69 ml/100 ml/min, an increase by 150±24% compared to baseline, which was in a steady-state after four min (Fig. 1). The calculated concentrations of both inulin and ICG corresponded with the concentrations measured in the venous plasma, at all time-points. Plasma concentrations of inulin and ICG measured in venous blood sampled from the contralateral arm at the end of the infusions, 6±1 mg/L and 0.6±0.2 mg/L, respectively, were negligible as compared to values measured in the infused arm.
3.2 Plasma concentrations at intermediate forearm blood flow
During the infusion with vehicle FBF remained constant (3.15±0.55 ml/100 ml/min). Again the calculated concentrations of inulin and ICG corresponded well with measured plasma concentrations, and plasma concentrations established in the contralateral arm at the end of the infusions were again negligible as compared to concentrations measured in the infused arm, 15±1 mg/l and 0.8±0.2 mg/l, respectively (see Fig. 2).
3.3 Plasma concentrations at low forearm blood flow
Methoxamine (1 µg/kg/min) significantly reduced FBF from 3.02±0.54 to 1.70±0.30 ml/100 ml/min, a decrease of 40±8% compared to baseline, which was at steady-state after four min (Fig. 3). The calculated concentrations of ICG still displayed a strong correspondence with the concentrations measured in the venous plasma, at all time-points (Fig. 3). The plasma concentrations of inulin did not fully reach the calculated values (P<0.05 at 4 and 8 min, respectively), although there were no significant differences between calculated and measured values at 16 min. Contralateral plasma concentrations of inulin and ICG measured at the end of the infusions, 13±1 mg/l and 0.6±0.3 mg/l, respectively, were negligible as compared to values measured in the infused arm.
3.4 Bland-Altman analysis
Bland-Altman analysis showed a high level of agreement between the calculated and measured plasma concentrations for both inulin and ICG. For inulin and ICG the differences in log-concentration were 0.131±0.449 and 0.045±0.609, respectively (mean±2 SD), and were not significantly different from nil.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Venous occlusion plethysmography of the forearm is widely used to indirectly assess the effects of intra-arterially infused vasoactive compounds on human peripheral resistance vessels (see [1, 2]). In order to relate drug concentrations in arteriolar plasma to pharmacologic effects the following has to be considered. Plasma concentrations of the infused compounds can provide important information on their pharmacologic characteristics. Substances that are continuously infused into the brachial artery reach the resistance vessels almost instantaneously. In addition, as long as these compounds have not yet passed through the capillary bed, they have not penetrated the extravascular tissue compartment. Since concentrations cannot be measured at the arteriolar level directly, they either have to be calculated or estimated by measurement in venous blood samples. The latter approach, however, is laborious and often limited by technical problems. Also, venous plasma concentrations may not be representative for the actual concentration that exists at the arteriolar side of the vascular bed, since infused compounds may diffuse into the extravascular compartment. In our present investigations, this issue is illustrated by the discrepancy in the measured plasma concentrations of inulin as compared to ICG.
ICG is a 775 dalton watersoluble tricarbocyanine dye that binds rapidly to plasma protein, of which albumin is the principle carrier (95%). It is taken up from the plasma almost exclusively by the hepatic parenchymal cells and is subsequently secreted entirely into the bile. ICG is clinically used for determining blood volume [10], cardiac output [15], hepatic function, liver and renal blood flow [16–18], blood flow through mesenteric and peripheral arteries [19, 20]and hemodialysis fistulae [21]. In our experiments, the ICG plasma concentration calculated corresponded strongly with the concentrations measured in venous blood samples, at all time-points and at all blood flow levels. This can be explained by the fact that most of the ICG binds to plasma protein almost immediately after it has been infused, so that it is greatly inhibited to leave the vascular compartment.
Inulin is an inert 5200 dalton uncharged polymer of fructose, which is clinically used for the determination of renal function by measurement of the glomerular filtration rate [22]. In contrast to ICG, inulin does not bind to plasma proteins and therefore can easily penetrate into the interstitial compartment when passing through the capillary bed. Therefore, its volume of distribution practically equals that of the extracellular compartment. Since the capillary extraction of inulin is rather flow independent (see ref. [11]) the measured plasma concentration in the venous effluent depends on the volume of distribution and the clearance rate. In an organ system, the latter is determined by the product of blood flow and the extraction rate [23]. Therefore, with a constant infusion rate at low forearm blood flows it will take longer to reach equilibrium between the plasma and tissue compartments, than at high forearm blood flows.
Our data are the first to show that over a wide range of flow conditions plasma concentrations of compounds infused intra-arterially into the vascular bed of the human forearm can be accurately estimated by calculation. The currently proposed approach, however, does not account for nonspecific loss of infused vasoactive compounds such as plasma protein binding, uptake or degradation mechanisms. The equation used provides an appropriate approximation of the total plasma concentrations of intra-arterially infused drugs at the level of the arterioles, considering the influence of variations in FBF. Furthermore, it indeed allows quantitative assessment of the pharmacologic characteristics of vasoactive compounds [6–9], and the characterization of receptors by classical pharmacologic techniques, as we have demonstrated for muscarinic receptor subtypes [6, 8].
Time for primary review 40 days.
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