Effects of protein binding on the placental transfer of propofol in the human dually perfused cotyledon in vitro

Y.-L. He*, S. Tsujimoto, M. Tanimoto, R. Okutani, K. Murakawa and C. Tashiro

Department of Anaesthesiology, Hyogo College of Medicine, 1–1 Mukogawa-cho, Nishinomiya City 663-8501, Japan

Accepted for publication: January 31, 2000


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The placental transfer of propofol was investigated using the in vitro dually perfused cotyledon model of the human placenta, and the effects of protein binding in the foetal perfusate were examined. Both maternal and foetal circulations were perfused in a single-pass mode and >30 min of stabilization was allowed before adding propofol and antipyrine to the maternal perfusate. The placental clearances of propofol were significantly increased by the augmented albumin concentrations in the foetal perfusate (1.68 (SD 0.68), 3.08 (1.55), 4.79 (1.76), 5.75 (1.89) and 7.03 (1.46) ml h–1 g–1 at the albumin concentrations of 4.4, 11, 22, 33 and 44 g litre–1, respectively). Although the total propofol concentration in the foetal vein increased significantly with increasing albumin concentration, the concentration of free propofol remained unchanged. These results indicate that binding to foetal albumin is a determining feature in the control of the placental transfer of propofol, and that the pharmacological effects of propofol on the foetus can be expected to be fairly constant and predictable from the maternal propofol concentration.

Br J Anaesth 2000; 84: 281–6

Keywords: anaesthetics, propofol; anaesthesia, obstetrics


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Propofol is becoming increasingly popular in clinical anaesthesia and the sedation of intensive care patients because of its rapid onset, short duration of action, and rapid recovery with a low incidence of postoperative side-effects. Several studies have been performed to evaluate the value and safety of propofol as an induction agent for general anaesthesia in comparison with thiopental in parturients undergoing caesarean section19 and for the maintenance of anaesthesia.8 9 Opinions on the safety of using propofol in parturients undergoing Caesarean section are divided. Although Celleno and colleagues reported that neonates from mothers who received propofol (2.4–2.8 mg kg–1) had muscular hypotonus, lower Apgar and neurobehavioural scores than those who received thiopental,3 6 six other centres concluded that propofol (1.5–2.5 mg kg–1) is a suitable alternative to thiopental.1 2 4 5 79 The foetal/maternal ratio of propofol at delivery was reported to be ~0.7,911 demonstrating that propofol diffuses across the placenta efficiently. Lipid solubility, the degree of ionization and the amount of protein binding are considered to influence the placental permeability of a drug. Propofol is a highly lipophilic compound and is extensively bound to plasma proteins (97–99%),1214 and drugs of this type are generally believed to bind to albumin in plasma. Therefore, the changes in concentrations of proteins such as albumin may have considerable effects on placental permeability and unbound drug, which is the pharmacologically active form. There are many practical difficulties in studying the factors governing the placental transfer of drugs in parturients. The in vitro perfused human placental preparation has been shown to be a useful model1521 for the accurate examination of the determinant(s) of the placental transfer of a drug, as it allows perfusate components to be manipulated without concern for the mother or foetus. The object of the present study was to investigate the placental transfer of propofol and the effects of protein binding in the foetal perfusate using the dually perfused human placental model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Placental perfusion
This study was approved by our institutional review board. Placentas from healthy parturients were obtained after Caesarean section and transported immediately to the laboratory. Foetal and maternal sides of the placenta were perfused following the method used in the in vitro perfusion model reported previously.22 An intact placental lobule was selected by inspecting the maternal side, and a chorionic artery and vein supplying a single peripheral cotyledon were cannulated. Figure 1 shows a schematic diagram of the perfusion system. The foetal circuit was perfused at the rate of 2.0 ml min–1. The cotyledon was then mounted in a specially designed chamber maintained at 37°C in a water bath, with the foetal surface of the placenta facing downwards. Three 20 G needles were gently placed in the decidual plate and the intervillous space was perfused at 15 ml min–1. Both foetal and maternal circuits were perfused in single-pass mode with tissue culture medium 199 modified with Earl’s salts (ICN Biomedicals, Aurora, OH, USA) containing heparin 2500 u litre–1, gentamicin 50 mg litre–1 and glucose 1.0 g litre–1. Human serum albumin (HSA) (plasma protein fraction; Baxter, Tokyo, Japan) was added to obtain desired HSA concentrations, and dextran (mol.wt 40 000; Wako Pure Chemicals, Osaka, Japan) was added to adjust the oncotic pressure. Sodium bicarbonate was added to keep the pH of the perfusate within the physiological range. Foetal and maternal perfusion pressures were measured with a sphygmomanometer (Yamasu, Tokyo, Japan). After a stabilization period of >30 min, propofol (Diprivan; Zeneca Pharmaceuticals, Osaka, Japan) and antipyrine (Wako Pure Chemicals, Osaka, Japan) were added to the maternal perfusate. The placental experiment was initiated at the albumin concentration of 4.4 g litre–1 in both circuits and returned to the same condition at the end of the perfusion experiment. In the intervening period, the albumin concentrations in foetal perfusate were varied at random among 11, 22, 33 and 44 g litre–1 at 30-min intervals, while the albumin concentration in the maternal perfusate was maintained at 4.4 g litre–1. Eight placentas were successfully perfused and three or four clearance estimations with different albumin concentrations in the foetal perfusate were performed in each placental preparation depending on tissue viability. Arterial and venous samples from the maternal circuit and venous samples from the foetal circuit were taken at 20 and 30 min during each experimental period for propofol and antipyrine measurement. A pilot single-pass perfusion experiment with foetal venous samples taken at 2, 5, 10, 15, 20 and 30 min demonstrated that propofol and antipyrine concentrations reached a plateau by 10 min. To measure the protein binding of propofol at different albumin concentrations, venous samples from the foetal circuit were collected during periods of 20–30 min. Venous samples were also taken from the foetal and maternal circuits at 30-min intervals for the measurement of human chorionic gonadotropin (hCG) concentration. The synthesis and release of hCG by the syncytiotrophoblast is a direct indication of the metabolic activity of the perfused cotyledon. Additional arterial and venous samples were taken for the measurement of glucose and lactate concentrations, to estimate glucose consumption and lactate production. At the end of the experiment, perfused placental tissue was dissected and weighed, and the concentrations of propofol and antipyrine were determined.



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Fig 1 Schematic representation of the experimental setup for perfusing a human placental cotyledon in vitro.

 
Sample analysis
Propofol and antipyrine were measured using modified high-performance liquid chromatography (HPLC) methods reported previously by Plummer23 and Ha et al.,24 respectively. The HPLC apparatus (LC-10AD; Shimadzu, Kyoto, Japan) comprised a variable-wavelength UV detector (SPD-10AD; Shimadzu) and fluorimetric detector (RF-10AD; Shimadzu), a degasser (DGU-14A; Shimadzu), an autoinjector (SIL-10AD; Shimadzu) and a column oven (CTO-10AS; Shimadzu). Briefly, a Symmetry C18 column (3.5 µm, 4.6x100 mm; Waters, Japan) was used for propofol analysis with fluorometric detection. For antipyrine, a Nova-PakR C18 column (4.0 µm, 3.9x150 mm; Waters) was used with the UV detector set at 210 nm. Placental tissue homogenate (10%) was prepared with the mobile phase for propofol measurement. Both perfusate and tissue homogenate samples were deproteinized with 10 volumes of acetonitrile, and 10 µl of the supernatant was injected onto the chromatograph. The intra- and inter-assay variabilities for propofol and antipyrine were <5% over the concentration range studied. The perfusate concentrations of hCG were measured by enzyme immunoassay using an IMxTM hCG Dinapack (Dainabot, Tokyo, Japan). Glucose and lactate concentrations were measured using a blood gas, electrolyte, metabolite analyser (ABL system 625; Radiometer, Copenhagen, Denmark).

Measurement of propofol binding
The unbound fraction of propofol in the perfusate samples was determined by an ultrafiltration method using the CentrifreeTM Micropartition System (Amicon, Bedford, MA, USA).

Calculations of clearance and clearance index
The placental transfer of propofol and antipyrine was evaluated as transplacental clearance. A single-pass experimental design was used for both maternal and foetal circulations. The transplacental clearance was then calculated as follows:22


where Qf is the flow rate of the foetal circulation, Cfv and Cma are the venous concentration in the foetal circuit and the arterial concentration in the maternal circuit, respectively. Transplacental clearance was estimated at 20 and 30 min in each perfusion period at different albumin concentrations in the foetal perfusate. The clearance index (CI) of propofol was calculated based on the transplacental clearances of propofol (CLpropofol) and antipyrine (CLantipyrine), as follows:


Statistics
All data are expressed as mean (SD). SigmaStat for Windows (Version 1.0; Jandel Scientific, Chicago, IL, USA) was used for the statistical analysis. The propofol concentrations, transplacental clearances and clearance indexes were analysed by one-way repeated measures analysis of variance. If the analysis of variance was found to be significant, Bonferroni’s test was performed to compare the values at various albumin concentrations in the foetal perfusate with that at 4.4 g litre–1. The significance of the difference between placental tissue and maternal venous concentrations of propofol was evaluated with the paired t-test. Differences were considered to be significant when P<0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The characteristics of the perfused cotyledons are summarized in Table 1. Glucose consumption, lactate production and the production of hCG showed values similar to those reported previously,2526 indicating the physiological integrity of the perfused cotyledons. No hCG was detected in the foetal circulation, suggesting the expected preferential secretion of the hormone and the physical integrity of the placental preparation.


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Table 1 Characteristics of the perfused human placentas. All data are expressed as mean (SD)
 
Propofol was detected in foetal samples within 2 min, suggesting that propofol passes across the placenta rapidly. The propofol concentrations in the maternal artery, vein and foetal vein at various albumin concentrations in the foetal perfusate are illustrated in Figure 2. The foetal venous propofol concentrations increased significantly as the albumin concentration increased from 4.4 to 11, 22, 33 and 44 g litre–1 (P<0.05), while the maternal venous propofol concentration remained unchanged. The propofol concentration in the foetal vein increased to the level found in maternal circulation when the albumin concentration in the foetal perfusate was maintained at the physiological level of 44 g litre–1, resulting in a foetal/maternal concentration ratio of 1.13 (SD 0.15). In contrast, the concentration of free propofol in the foetal vein was not significantly affected by the albumin concentration (Fig. 2).



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Fig 2 Propofol concentrations in maternal artery (diamond), maternal vein (down filled triangle), foetal vein (filled circle), and the unbound foetal venous concentrations (up filled triangle) at various concentrations of human serum albumin in the foetal perfusate.

 
Figure 3 illustrates the effects of albumin concentration in the foetal circulation on the transplacental clearances of propofol and antipyrine and the clearance indexes. The placental clearance of antipyrine, which is commonly used as a reference compound in placental perfusion, did not change significantly at a variety of albumin concentrations over the range of 3.23 (SD 1.1) to 3.90 (1.40) ml h–1 g–1 (Fig. 3). The placental clearance of propofol was 1.68 (0.68) ml h–1 g–1 at the albumin concentration of 4.4 g litre–1, with a clearance index of 0.51 (0.09). It was significantly increased to 3.08 (1.55), 4.79 (1.76), 5.75 (1.89) and 7.03 (1.46) ml h–1 g–1 by increasing the albumin concentrations to 11, 22, 33 and 44 g litre–1, respectively (P<0.05). Consequently, the clearance index exceeded unity when the albumin concentration was >=22 g litre–1 (0.76 (0.20), 1.29 (0.19), 1.49 (0.21) and 2.01 (0.40) at albumin concentrations of 11, 22, 33 and 44 g litre–1, respectively). The mean propofol concentration in the perfused placental tissue at the end of the experiments was 53.3 (20) µg g–1 wet tissue, which was significantly higher than the corresponding mean maternal venous concentration (9.7 (0.9) µg ml–1; P<0.001).



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Fig 3 Effects of human serum albumin concentrations in the foetal perfusate on the transplacental clearances of propofol (open columns), antipyrine (hatched columns) and clearance indexes (cross-hatched columns).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In obstetric anaesthesia, the concern has generally been to minimize foetal exposure, but the foetus has now also become the object of intended drug treatment,27 which is usually administered via the maternal circulation. Knowledge about the placental transfer and uptake of anaesthetics and the factors that may influence transplacental passage should gain increasing therapeutic importance in obstetric anaesthesia for foetal therapy. The commonly used foetal/maternal concentration ratio varies, depending on factors such as the interval between drug administration and delivery, and the protein binding of a drug in maternal and foetal plasma.28 Thiopental, which is the most commonly used induction agent in obstetrics, crosses the placenta rapidly and can be detected in umbilical blood within 30 s of its administration to the mother.29 The mean foetal/maternal ratio for thiopental has been reported to be in the range of 0.4–1.0.30 Albumin concentrations in both mother and foetus are reported to show dynamic changes during pregnancy.31 The maternal and foetal albumin concentrations were shown to range between 25 and 35 g litre–1 and between 7.5 and 39.8 g litre–1, respectively, over weeks 12–40 of gestation.31 In this study, the placental transfer of propofol was investigated using the perfused human placental model, which allowed us to simulate this in vivo situation by varying the albumin concentration in the foetal circulation from 4.4 to 44 g litre–1.

Antipyrine serves as an ideal marker of placental transfer because it is of intermediate solubility and does not bind to plasma proteins. The placental clearance of antipyrine remained unchanged throughout the experimental period at different albumin concentrations in the foetal perfusate (Fig. 3), suggesting that the transplacental exchange of small molecules was maintained throughout the perfusion period. The transplacental clearance of propofol increased considerably with increasing albumin concentration, and was significantly greater than that of antipyrine when the albumin concentration in the foetal circulation was >=22 g litre–1. This result indicates that the placental permeability of propofol might be more efficient than that for antipyrine during the pregnancy period close to term. Increasing the albumin concentration in the foetal perfusate led to a significant increase in the placental transfer rate of propofol, demonstrating that binding to albumin in the foetal circulation is a dominant feature in the control of the placental transfer of propofol. Binding of propofol to plasma was reported to be independent of the substrate concentration over the range 0.04–150 mg ml–1;32 therefore, the binding capacity for propofol is expected to be proportional to the albumin concentration according to the formula B/F = nKaP, where B is the bound drug concentration, F is the unbound drug concentration, n is the number of binding sites per molecule of protein, Ka is the affinity constant and P is the concentration of binding protein.33 Consistent with this suggestion, the observed unbound fractions of propofol in foetal venous samples were 16.9 (SD 3.6), 12.7 (3.3), 6.8 (1.5), 4.2 (1.1) and 2.8 (0.5)% at albumin concentrations of 4.4, 11, 22, 33 and 44 g litre–1, respectively. Therefore, the facilitated placental transfer of propofol can be readily explained by the differences in protein-binding capacity at various albumin concentrations, because the increased protein-binding capacity for propofol at higher albumin concentrations in the foetal perfusate allows a larger amount of the drug to cross the placenta faster. This is consistent with observations using the same perfused human placenta model with several other lipophilic drugs, including thiopental, demonstrating that transplacental distribution was markedly influenced by maternal and foetal protein capacity.17 20 3437

The placenta is unique in being perfused on both its maternal and its foetal surface, and foetal blood is separated from that of the mother by the placental trophoblast and the foetal capillary system. Therefore, the transplacental exchange of a drug comprises three processes: distribution from the maternal circulation to the placenta; placental sequestration, including intracellular binding to placental component(s) and placental metabolism; and transfer out of the placenta to the foetal and maternal circulations. The maternal venous concentrations were not influenced by the albumin concentration in the foetal perfusate, resulting in constant extraction of propofol from the maternal circulation, which was about 15% (Fig. 2). Therefore the considerable facilitation of the transplacental exchange of propofol by increased albumin concentrations can be attributed to the facilitated placental sequestration of propofol from the placenta to the foetal circulation. That is, the placental transfer of propofol is restricted by placental retention at lower albumin concentrations in the foetal perfusate. Consistent with this, high placental tissue concentrations of propofol (53.3 (20) µg ml–1) were observed at the end of the experiment. This implies that the placenta plays an important role in protecting the foetus from exposure to propofol administered to the mother as a depot. On the basis of a simple model that has been proposed to interpret the effects of binding to serum albumin and to the placenta on the placental transfer of a drug,35 the protein-binding capacity and the relative affinity of propofol for maternal and foetal plasma proteins and to placental component(s) can be considered to be the predominant features of the control of transfer across the placenta. Furthermore, restricted foetal transfer associated with pronounced placental accumulation for lipophilic drugs, such as bupivacaine, dexmedetomidine and sufentanil, has also been reported.17 18 20 21

The foetal/maternal concentration ratio of propofol varied with the albumin concentration in the foetal perfusate, ranging from 0.74 (0.28) to 1.13 (0.15) over the albumin concentration range of 22–44 g litre–1 in this human perfused cotyledon model. This indicates that the clinically measured foetal/maternal ratio, which is considered to be an index of the placental transfer of a drug, can show a wide range depending on the plasma protein concentrations. It is of note that the free concentrations of propofol in the foetal perfusate did not change significantly, irrespective of the considerably increased total propofol concentrations (Fig. 2). Therefore, the pharmacological effects of propofol will not be influenced by the plasma albumin concentration in the foetus. However, in vivo, foetal uptake of propofol is a dynamic process, and on the basis of the lower umbilical arterial concentrations compared with those in umbilical vein samples it has been suggested that the neonate continues to take up propofol during delivery, a process that may involve neonatal metabolism.9 Protein binding can be seen as a mechanism whereby propofol is transported to and from the peripheral tissues; therefore, the storage function of plasma protein binding should not be neglected. The enormously increased bound component in the foetus at higher albumin concentrations might result in a prolonged effect of propofol on the foetus, and more importantly, on the neonate after birth.

In summary, placental transfer of propofol was significantly facilitated by increased albumin concentrations in the foetal circulation, indicating that the binding capacity to albumin in the foetal circulation is a dominant feature in the control of the transplacental exchange. The high propofol concentration in placental tissue demonstrates that the placenta plays an important role in protecting the foetus from exposure to propofol administered to the mother as a depot. The free concentration of propofol did not change significantly despite the considerably increase in total propofol concentration in the foetal circulation associated with the increased albumin concentration, suggesting that the pharmacological effect of propofol on the foetus can be expected to be fairly constant and predictable from the maternal propofol concentration. However, the storage function of plasma protein binding should not be neglected.


    Acknowledgements
 
This study was supported by Grant-in-Aid B No. 11470330 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to C.T.


    Footnotes
 
* Corresponding author Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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