Total Propofol Concentrations Research Articles

Binding of Propofol to Human Serum Albumin

The binding properties of the short-acting hypnotic agent propofol (CAS 2078-54-8) to human serum albumin were studied in vitro. Using centrifugation through ultrafiltration membranes the ratio of free and bound propofol as a function of the human serum albumin concentration was determined. In addition, a biomathematical approach was tested that allowed the determination of the number of binding sites from the measured binding profile. At a total propofol concentration of 8.81 +/- 0.25 microg/mL the concentration of the free fraction of propofol ranged from 338 +/- 11 ng/mL (mean +/- SE) at an albumin concentration of 0.5% to 15 +/- 2 ng/ mL at an albumin concentration of 8.0%. The corresponding percentage of propofol binding ranged from 96.07 +/- 0.14% to 99.83 +/- 0.02%. From this binding profile the binding site was estimated to be n(0) = 2. The measured influence of the albumin concentration on propofol binding might be of relevance in states of hypo- or hyperalbuminaemia which could result in an altered demand of propofol.

Anomalous relationship between free drug fraction and its total concentration in drug–protein systems: II. Binding of different ligands to plasma proteins

As it was reported earlier, the free propofol percentage is dependent on its total concentration in human blood or plasma protein solution. At total propofol concentrations below 1 μg ml −1 the free fraction of the drug increases with the decrease of its total concentration. The results of the experiments performed in Part I indicate that the well-ordered water around a protein molecule (i.e. the hydration layer) is probably the main factor responsible for the mentioned anomalous free drug fraction changes. The experiments carried out in this study with different ligands (homologous saturated fatty acids, indomethacin and lidocaine) show that: 1. ligand hydrophobicity affects the run of the dependence between the free ligand fraction and its total concentration, which confirms the importance of the hydration layer in the drug binding process, 2. similar anomalous changes of the free drug fraction are observed not only for drugs interacting with different binding sites on human serum albumin (HSA), i.e. for propofol and indomethacin, but also for basic local anesthetic lidocaine which is known to bind to other plasma protein, alpha-1-acid glycoprotein (AGP). Therefore, the increase of the free drug fraction at its low total concentration does not appear to be an exclusive feature of propofol–HSA system. The results presented in this paper expand the present knowledge about drug–protein binding process and thereby encourage further research on this subject.

Anomalous relationship between free drug fraction and its total concentration in drug–protein systems: I. Investigation of propofol binding in model HSA solution

According to contemporary knowledge, only the free form of a drug exhibits pharmacological activity due to its ability to cross the membranes and to interact with receptors. For the majority of drugs their free fraction in human plasma is usually constant, hence the therapeutic effect of a drug is most often correlated with its total concentration. However, in the case of certain drugs of narrow therapeutic window, for which a slight elevation of a free drug fraction (e.g., occurring in some pathological states) may exert a toxic effect, the therapeutic effect should be correlated with free concentration. As we reported earlier, at total propofol concentrations below 1 μg ml −1 the free fraction of the drug increases with the decrease of its total concentration. The presented paper discusses the results of several experiments performed in order to elucidate this anomalous phenomenon. The study is mainly focused on the investigation of the relationship between free drug percentage and total drug concentration in human serum albumin (HSA) solution, performed at different temperatures and in the presence of the salting-out agent. It was observed that a temperature increase leads to an increased propofol binding, which confirms the importance of hydrophobic interactions for the binding process. The addition of the salting-out agent significantly changing water structure of the drug–protein system is followed by disappearance of the anomalous relationship and results in constant value of the free drug fraction in the whole range of the studied total drug concentration. The results of the performed experiments indicate that the well-ordered water around a protein molecule (i.e., the hydration layer) constitutes the main factor responsible for the discussed anomalous free drug fraction changes.

Propofol enhances both tonic and phasic inhibitory currents in second-order neurons of the solitary tract nucleus (NTS)

The anesthetic propofol is thought to induce rapid hypnotic sedation by facilitating a GABAergic tonic current in forebrain neurons. The depression of cardiovascular and respiratory regulation often observed during propofol suggests potential additional actions within the brainstem. Here we determined the impacts of propofol on both GABAergic and glutamatergic synaptic mechanisms in a class of solitary tract nucleus (NTS) neurons common to brainstem reflex pathways. In horizontal brainstem slices, we recorded from NTS neurons directly activated by solitary tract (ST) axons. We identified these second-order NTS neurons by time-invariant (“jitter” < 200 μs), “all-or-none” glutamatergic excitatory postsynaptic currents (EPSCs) in response to shocks to the ST. In order to assess propofol actions, we measured ST-evoked, spontaneous and miniature EPSCs and inhibitory postsynaptic currents (IPSCs) during propofol exposure. Propofol prolonged miniature IPSC decay time constants by 50% above control at 1.8 μM. Low concentrations of gabazine (SR-95531) blocked phasic GABA currents. At higher concentrations, propofol (30 μM) induced a gabazine-insensitive tonic current that was blocked by picrotoxin or bicuculline. In contrast, total propofol concentrations up to 30 μM had no effect on EPSCs. Thus, propofol enhanced phasic GABA events in NTS at lower concentrations than tonic current induction, opposite to the relative sensitivity observed in forebrain regions. These data suggest that therapeutic levels of propofol facilitate phasic (synaptic) inhibitory transmission in second-order NTS neurons which likely inhibits autonomic reflex pathways during anesthesia.

The Increase in Total and Unbound Propofol Concentrations During Accidental Hemorrhagic Shock in Patients Undergoing Liver Transplantation

To the Editor: Hemorrhagic shock increases the sedative effects of propofol (1,2), so that a patient in hemorrhagic shock should receive only 10%–20% of the propofol dose that a healthy patient would receive (3). However, the mechanism of increase of end-organ sensitivity remains unclear. We demonstrate increases in total and unbound propofol in four patients with advanced cirrhosis who became hemodynamically unstable as a result of massive blood loss during surgery. After IRB approval and with written informed consent from patient, we induced anesthesia with vecuronium 0.1 mg/kg, and propofol 2 mg/kg and maintained anesthesia with 60% air, 0.5%–1.5% isoflurane in oxygen, fentanyl 30 g/kg, and propofol infusion of 2 mg/kg/h. After propofol injection, we collected arterial blood samples to measure blood propofol concentration. We used high-performance liquid chromatography to measure total and unbound propofol concentrations, as reported previously (4). Table 1 shows systolic arterial blood pressure, heart rate, total propofol, fraction unbound, and unbound propofol. After hemorrhagic shock, all patients were infused with lactated Ringer’s solution and mannitol adenine phosphate or were administered dopamine. All patients were adequately resuscitated within 60 min. Total propofol concentrations increased two-fold during hemorrhagic shock, as did the unbound fraction of propofol. The unbound propofol concentrations increased four-fold during hemorrhagic shock.Table 1: Hemodynamic Data and Propofol ConcentrationsThe total propofol concentration likely increased because of the physiological response to shock in our patients. Specifically, shock likely resulted in substantial reductions in hepatic and renal blood flow. Because the clearance of propofol is highly sensitive to liver and renal blood flow (4,5), the propofol clearance decreased during shock, increasing the concentrations during the infusion. This is consistent with effects of experimental hemorrhagic shock on total propofol concentrations in an animal study (1,2,6). However, relatively little attention has been given to hemorrhagic shock’s effect on unbound propofol concentrations. Propofol is a drug with a large distribution volume, high total body clearance (7), and a high degree of binding to plasma albumin, erythrocytes and lipoproteins (8,9) (unbound fraction of <3%). Therefore, the change of unbound fraction is clinically important as we reported previously (3,10). The unbound drug concentration is responsible for a drug’s pharmacological effects, as it is the unbound concentration that equilibrates between the blood and tissues. As the unbound propofol increased four-fold in our patients, this implies that the propofol dose requirements for these patients could be decreased by about 75%, consistent with Shafer’s analysis (3). Daisuke Takizawa, MD Department of Anesthesiology Saitama Cardiovascular and Pulmonary Center Osato-gun, Saitama 360-0105, Japan Department of Anesthesiology Gunma University Graduate School of Medicine 3-39-22 Showa-machi, Maebashi 371-8511, Japan [email protected] Eri Takizawa, MD Department of Anesthesiology Gunma University Graduate School of Medicine 3-39-22 Showa-machi, Maebashi 371-8511, Japan Sohtaro Miyoshi, PhD Fuminori Kawahara, PhD Haruhiko Hiraoka, PhD Department of Anesthesiology Saitama Cardiovascular and Pulmonary Center Osato-gun, Saitama 360-0105, Japan

Disposition and pharmacodynamics of propofol during isovolaemic haemorrhage followed by crystalloid resuscitation in humans

The purpose of this study was to estimate the changes in unbound propofol concentration and pharmacodynamics of propofol during isovolaemic haemorrhage followed by crystalloid resuscitation. Ten patients undergoing measure elective surgery were enrolled in this study. Anaesthesia was maintained by 60% nitrous oxide in oxygen, fentanyl 10-20 microg kg-1 and an infusion of propofol at 8 mg kg-1 h-1 until the end of the operation. Radial arterial samples were collected for measurement of propofol concentration just before the start of the operation, and at the point when blood loss was >10 ml kg-1, 20 ml kg-1 and 30 ml kg-1. Cardiac output (CO), haemoglobin values and plasma concentrations of albumin were also determined. Patients were resuscitated with lactated Ringer's solution to maintain a mean arterial blood pressure (+/-20% of prehaemorrhage). Bispectral index (BIS) was measured continuously. Mean blood pressure, heart rate and CO were well maintained during the operation in all patients. Haemoglobin values and plasma albumin concentrations decreased significantly during surgery. There were no significant differences in total propofol concentrations across the time points. The unbound propofol concentration was increased from 0.10+/-0.040 microg ml-1 to 0.17+/-0.041 microg ml-1 after the haemorrhage of 30 ml kg-1 (P<0.05). BIS was significantly decreased from 47+/-5.9 to 39+/-3.7 (P<0.05) after the haemorrhage of 30 ml kg-1. The hypnotic potency of propofol is increased during isovolaemic haemorrhage in crystalloid resuscitated patients even if CO is maintained.

Influence of propofol concentration in human plasma on free fraction of the drug

Drugs exist in blood in two forms: free and bound to proteins and blood cells. It is generally assumed that only the unbound form of a drug exerts pharmacological activity as it is able to diffuse across the membranes and reach the site of action. Since for the majority of drugs their free fraction is usually constant, the therapeutic effect of the drug is most often correlated with its total concentration. However, in case of some disease states (e.g. renal or hepatic disorders) the protein concentration may change dramatically, resulting in clinically significant change of free drug fraction. The results presented in the paper prove that, in case of propofol, an increase of free fraction occurs with a decrease of total drug concentration. This dependence is observed both in vitro (in artificial and native human plasma) and in vivo. Free propofol fraction, which in clinical conditions ranges from 1 to 3%, at very low total propofol concentrations (below 0.01 μg ml −1) tends to reach 100%. This increase of free drug percentage is discussed in terms of its possible reasons as well as its potential clinical relevance.

Changes in drug plasma concentrations of an extensively bound and highly extracted drug, propofol, in response to altered plasma binding

Cardiopulmonary bypass is known to result in a reduction in the plasma binding of drugs. The resulting effect on the hepatic clearance of drugs with low extraction is well understood. However, the situation with those that are highly extracted is less clear. Studies were, therefore, undertaken with one such drug, propofol, for which plasma binding was changed during cardiac surgery with cardiopulmonary bypass. After induction of anesthesia with midazolam in 19 patients, propofol was infused continuously at a rate of 4 mg. kg(-1). h(-1) during surgery. Propofol's concentration was measured by HPLC in blood samples collected from the radial artery and hepatic vein during surgery at predetermined intervals. The drug's unbound fraction in arterial plasma was estimated via equilibrium dialysis. The total concentration of propofol in blood was unchanged during surgery except shortly after the initiation of cardiopulmonary bypass. By contrast, the fraction of unbound propofol in blood increased by 2-fold during cardiopulmonary bypass and then decreased after the completion of cardiopulmonary bypass. The hepatic extraction ratio of propofol was greater than 0.8 and remained constant throughout surgery. The ratio of propofol concentration in erythrocytes to that in blood increased by 1.6-fold during cardiopulmonary bypass. During cardiopulmonary bypass, a significant increase in the concentration of unbound propofol occurred without alteration in the total propofol concentration in blood. The effect of the changes of propofol's protein binding on its kinetics was consistent with the predictions based on the well-stirred model of hepatic elimination for an intravenously infused high-clearance drug. Our finding on propofol pharmacokinetics may be the first example demonstrating the theoretic prediction of the well-stirred model.

HPLC investigation of free and bound propofol in human plasma and cerebrospinal fluid.

The paper compares the total propofol concentration in the cerebrospinal fluid (CSF) with the free drug concentration in plasma measured in 35 humans scheduled for elective neurosurgical procedures during propofol anaesthesia. The concentrations of total and free propofol in the blood and CSF samples were measured by means of HPLC using liquid-liquid extraction and ultrafiltration in the sample preparation procedure. The arterial blood and CSF samples (collected from intraventricular drainage) were taken at the same time. According to the obtained results, the usually expected equality between free drug concentration in plasma and its total concentration in CSF is not valid for propofol: the unbound propofol concentration in plasma is not equal to its total concentration in CSF (p < 0.05). This difference suggests a substantial contribution of active transport in propofol transfer from blood into CSF. Moreover, the paper shows the presence of bound propofol in CSF, which is a novel finding.

Free and bound propofol concentrations in human cerebrospinal fluid.

The aim of this study was to define the relationship between unbound propofol concentrations in plasma and total drug concentrations in human cerebrospinal fluid (CSF), and to determine whether propofol exists in the CSF in bound form. Forty-three patients (divided into three groups) scheduled for elective intracranial procedures and anaesthetized by propofol target control infusion (TCI) were studied. Blood and CSF samples (taken from the radial artery, and the intraventricular drainage, respectively) from group I (17 patients) were used to investigate the relationship between unbound propofol concentration in plasma and total concentration of the drug in CSF. CSF samples taken from group II (18 patients) were used to confirm the presence of the bound form of propofol in this fluid. The CSF and blood samples taken from group III (eight patients) were used to monitor the course of free and bound CSF propofol concentrations during anaesthesia. For group I patients the mean (and 95% confidence interval) total plasma propofol concentration was 6113 (4971, 7255) ng ml(-1), the mean free propofol concentration in plasma was 63 (42, 84) ng ml(-1), and the mean total propofol concentration in CSF was 96 (76, 116) ng ml(-1) (P < 0.05 for the difference between the last two values). For group II patients the fraction of free propofol in CSF was 31 (26, 37)%. For group III patients the fraction of free propofol in CSF during TCI was almost constant (about 36%). The unbound propofol concentration in plasma was not equal to its total concentration in CSF and cannot be directly related to the drug concentration in the brain. Binding of propofol to components of the CSF may be an additional mechanism regulating the transport of the drug from blood into CSF.

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

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.

Cardiopulmonary bypass-induced changes in plasma concentrations of propofol and in auditory evoked potentials

Unbound, rather than total, plasma concentrations may be related to the anaesthetic action of propofol. Therefore, we measured plasma concentrations of propofol and recorded Nb wave latencies of auditory evoked potentials (AEP) during continuous infusion of propofol in 15 patients undergoing coronary artery bypass grafting (CABG) surgery. After induction of anaesthesia with fentanyl, propofol was infused continuously at a rate of 10 mg kg-1 h-1 for 20 min, and then the rate was reduced to 3 mg kg-1 h-1. Administration of heparin before cardiopulmonary bypass (CPB) did not affect total or unbound propofol concentration. Initiation of CPB decreased mean total propofol concentration from 2.6 to 1.7 micrograms ml-1 (P < 0.01). Simultaneously, mean unbound propofol concentration remained at 0.06 micrograms ml-1 because of a slight increase in the mean free fraction of plasma propofol (from 2.3 to 3.5%; P > 0.05). During hypothermic CPB, mean total propofol concentration increased to concentrations measured before bypass (to 2.1 micrograms ml-1; P > 0.05 vs value before CPB) and the mean unbound propofol concentration was at its highest (0.07 microgram ml-1; P < 0.05 vs value before heparin). After CPB and administration of protamine, the mean total propofol concentration remained lowered (1.7 micrograms ml-1; P < 0.05 vs value before heparin) and the mean unbound propofol concentration returned to the level measured before heparin (P < 0.001 vs value during hypothermia). The latency of the Nb wave from recordings of AEP increased after induction of anaesthesia, reached its maximum during hypothermia and was prolonged during the subsequent phases of the study. The latency of the Nb wave did not correlate with total or unbound propofol concentration. We conclude that the changes in total and unbound concentrations of plasma propofol were not parallel in patients undergoing CABG. During CPB or at any other time during the CABG procedure, the unbound propofol concentration did not decrease and Nb wave latency was prolonged compared with baseline values measured after induction of anaesthesia before the start of CPB.