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Introduction
The main medium distributing drugs in the human organism is blood, in which they are partly bound with proteins and
blood cells and partly exist in a free form. It is generally assumed that only an unbound drug can reach the site of action by
diffusing across the membranes and exerting pharmacological effects by interacting with
receptors[1]. For this reason, the therapeutic effect of a drug should be related not to its total, but to its free concentration. Most frequently, however, the
therapeutic effect of a drug is correlated with its total concentration, since for many drugs, their free fraction is constant in the
wide range of their total concentrations. Moreover, the procedure of total drug assay is cheaper, quicker and simpler. Free
drug measurement is recommended mainly for the potentially dangerous drugs of a narrow therapeutic window with
significant toxic effects at higher concentrations and when the protein level is
changing[2], for example, in the case of renal or hepatic
failure[3,4]. It should also be remembered that changes in the free fraction of a drug are more pronounced for highly-bound
drugs, where even a slight decrease in protein binding may substantially increase their free fraction and cause a dangerous
toxic effect.
Propofol is an example of a high (97%_99%) protein-binding
drug[5_9]. It is one of the most frequently applied intravenous
anesthetics used mainly for induction and maintenance of total anesthesia. It is also used in smaller doses for
sedative, antiemetic, antipruritic or anticonvulsant
purposes[10]. As its effect site is the central nervous system, propofol can be highly
dangerous when applied as a sedative agent in long-time infusions to high-risk patients hospitalized in intensive care units.
The decrease of total propofol concentration below 3
mg/mL leads to an increase of its free
fraction[11]. At very low total propofol concentrations, its free fraction tends to reach
100%[11]. The cited results were obtained when
investigating the total and free drug concentration in human plasma and human serum albumin (HSA) solutions spiked with an ethanolic solution
of propofol. It is not known however if the same happens in protein samples (plasma or HSA) spiked with propofol in
intralipid emulsion (eg Diprivan, Astra-Zeneca, Caponago, Italy). Some changes can be expected,
taking into account the results obtained by Zamacona
et al[12], who have shown the increase of propofol binding by blood proteins at elevated
concentrations of triglycerides and cholesterol. The resolution of this problem is important for 2 reasons: (1) clinically,
because in a typical clinical procedure propofol is administered in the form of soybean oil emulsion; and (2) analytically, to
find out if it is essential to spike protein samples with propofol in intralipid solution or if the ethanolic solutions of propofol
are sufficient to model the discussed phenomenon.
Materials and methods
Sample preparation and processing
Artificial plasma (HSA
solution)A part of the investigations was performed using artificial plasma solutions containing
different amounts of HSA. They were obtained by diluting the HSA preparation (ZLB, Bern, Switzerland) with the
physiological sodium chloride solution (Polfa, Lublin, Poland) to a required concentration. The following solutions were prepared: 3, 4,
6.5 and 8 g/100 mL.
Human plasma Plasma was obtained from the blood of a healthy volunteer. Blood sampling was performed after
obtaining an approval from the University Ethics Committee and consent from the subject. The plasma was isolated from
heparinized blood by centrifugation at
85×g.
Propofol solutionsThree types of propofol solutions were prepared:
1. Artificial plasma samples containing 4 g of HSA per 100 mL of the solution were spiked with propofol (Aldrich,
Steinheim, Germany) solution in 40% ethanol (POCh, Gliwice, Poland) to reach the following total propofol concentrations: 1,
3, 5, 8, and 12 mg/mL. No intralipid was present in these samples. The final concentration of ethanol in the samples was 0.2%
(different volumes of propofol solution were mixed with 40% ethanol in order to introduce the constant volume of alcohol into
each sample).
2. Artificial plasma samples containing 3 or 4 or 6.5 or
8 g HSA/100 mL were spiked with Diprivan™ preparation (AstraZeneca, Caponago, Italy), that is, 1% propofol solution in
intralipid to the following total propofol concentrations: 1, 3, 5, 8, and 12
mg/mL. The final samples contained the intralipid
concentration of 10, 30, 50, 80, and 120 mg/mL respectively.
3. Artificial plasma samples containing 5 g of HSA per 100 mL of the solution were spiked with Diprivan. Subsequently,
intralipid (100 mg/mL soybean oil/water emulsion; Baxter Healthcare, Thetford, UK) was added so that the final samples
contained constant amount of intralipid (120 mg/mL) and 1, 3, 5, 8, or 12
mg/mL of propofol.
Apart from the artificial systems, samples of human plasma spiked with intralipid propofol solution to the total propofol
concentrations of 1, 3, 5, 8, and 12 mg/mL were studied. The obtained samples contained the intralipid concentrations of 10,
30, 50, 80, and 120 mg/mL, respectively.
Unbound propofol isolation Unbound propofol was isolated by ultrafiltration on Amicon Micropartition System (MPS)
units (Millipore, Bedford, MA, USA), utilising the YM-10 membranes (product no. 40424, Millipore, USA) of a 10 kDa
molecular mass cut-off. The ultrafiltration units were centrifuged in a constant rotor angle centrifuge MPW-341 (Mechanika
Precyzyjna, Warsaw, Poland). One mL of each solution was put into the sample compartment of the ultrafiltration unit. After
the attachment of the ultrafiltrate collection container, the unit was centrifuged at
800×g until 400 mL of ultrafiltrate was
obtained.
Propofol assay For propofol assay, to each ultrafiltrate of plasma sample (artificial or native, 400
mL), thymol (internal standard), dihydrogen sodium phosphate (1 mL of 0.1 mol/L
NaH2PO4), and cyclohexane (3 mL) were added. The mixtures
were vigorously shaken for 10 min at 200 r/min. After centrifugation
1100×g for 5 min, to separate the phases, an aliquot of the
cyclohexane layer (2 mL) was transferred to a clean tube with tetramethylammonium hydroxide (TMAH) solution (10 µL). The
solvent was evaporated until dry in a stream of nitrogen. The residue was redissolved in the mobile phase and injected into
the chromatographic column. The limit of propofol quantitation in plasma ultrafiltrate was 3.7 ng/mL with a coefficient of
variation (n=3) of 11.1% at 5 ng/mL, 12.1% at 20 ng/mL and 9.8% at 40 ng/mL. The relative error of determination
(n=3) was 32.0% at 5 ng/mL, 9.3% at 20 ng/mL and -6.8% at 40 ng/mL. The linearity range of the whole analytical procedure was from 3
to 650 ng/mL. Other details of sample preparation and the measurement procedure are found in other
studies[7_9].
Chromatographic
equipment The concentrations of propofol were measured by high performance liquid
chromatography (HPLC) in plasma as well as in cerebrospinal fluid (CSF). A Gilson liquid chromatograph (Middleton, WI, USA)
consisting of a dual high-pressure pump (Model
122)integrated with a manometric module and a dynamic mixer was employed for
HPLC analysis. Propofol in the plasma ultrafiltrate was detected with a fluorescence detector (Jasco FP-920, Tokyo, Japan)
set at an excitation wavelength of 276 nm and at an emission wavelength of 310 nm. Chromatographic separations were
carried out using a 150×4.6 mm id
C18 silica gel column (Kromasil
C18, 5 µm, HiChrom, Novato, CA, USA) equipped with a 0.5
µm prefilter (Supelco, Bellefonte, PA, USA) and a guard column ODS
C18 (Alltech, Deerfield, IL, USA). The samples were
injected into the column by a Model 7125 injection valve from Rheodyne (Cotati, CA, USA).
Reagents and solutions All chemicals, except those separately mentioned, were obtained from the Polish Factory of
Chemical Reagents-POCh (Gliwice, Poland) and were of analytical grade. A mixture composed of 75% methanol and 25%
deionized Milli-Q water (v/v) was used as the mobile phase. Stock solutions of thymol and propofol in methanol
(1 mg/mL) were each prepared and stored at 4
oC. TMAH (25% solution in methanol; Aldrich, Germany) was diluted with
isopropanol (3:37 v/v).
Results and Discussion
As earlier outlined, the objective of the present study was to verify if the free propofol fraction increase at a low total drug
concentration observed in plasma and HSA solutions spiked with propofol in ethanolic
solution[11] occurs when propofol is introduced into protein samples in intralipid emulsion. The run of the relationships between the free propofol fraction and
total drug concentration shown in
Figure 1 proves that the free propofol fraction increases irrespective of the type of propofol solvent used. Moreover, a
significant increase of the free propofol fraction below the total propofol concentration of 3
mg/mL was observed. Thus, the increase of the free propofol fraction resulting from the decrease of total drug concentration occurs both in the presence of
ethanol and intralipid.
In the present research, the precise range of the free propofol fraction is not as important as the occurrence of its increase
at a low total drug concentration. Hence, it was decided to apply the typical procedure of propofol determination by a simpler
and more reliable HPLC with fluorescence
detection[13] rather than the GC procedure requiring propofol
derivatization[11].
As seen in Figure 1, the main difference between the investigated systems is the level of unbound propofol percentage.
Spiked HSA with propofol in intralipid solution lowers the free drug fraction in comparison with the samples spiked with
ethanolic solution. The observed decrease of the free drug percentage can be attributed to the lipoprotein modification of
HSA by soybean lipids resulting in higher affinity of the formed lipoproteins towards propofol.
Other experiments performed with different HSA concentrations in artificial plasma spiked with intralipid solution of
propofol (see Figure 2) not only confirm the increase of the free propofol fraction at a low total drug concentration for a wide
range of protein concentrations (3_8 g/100 mL), but also allow one to formulate the relationship between the free drug
percentage and the total protein concentration (see Figure 3). The observed decrease of the unbound propofol percentage
with the increase of protein content in the sample is logical considering the binding properties of HSA towards the drug.
The phenomenon of the free propofol fraction increase at low total drug concentrations in the samples spiked with
propofol in the intralipid emulsion is typical, not only for artificial plasma, but also for the samples of natural plasma (see
Figure 4). As discussed, the increase of the free propofol fraction is observed below 3 µg/mL of the total propofol concentration.
The samples prepared for the experiments illustrated in Figures 1_4 contained not only different amounts of propofol, but
also proportionally different amounts of intralipid. Such variations are typical for patients receiving intralipid only in the form
of propofol intralipid emulsions, that is, the greater the propofol infusion dose, the greater the amount of infused intralipid. It
can be said that the observed increase of free propofol fraction resulting from the decrease of the total drug concentration is
connected with the lowering intralipid concentration in the samples. In order to exclude the influence of intralipid
tion changes on the observed phenomenon, it was decided that the experiment be repeating and keeping the intralipid
concentration at a constant level of 120 µg/mL, which corresponds to the highest investigated concentration of propofol.
The results of this experiment are presented in Figure 5. The data in Figure 5 may be treated as more representative for the
samples taken from patients receiving not only propofol infusions, but also drug-free intralipid as a parenteral nutrient.
Figure 5 shows that the decrease of the total propofol concentration below 3 µg/mL
leads to an increase of its free fraction despite the intralipid concentration. The smaller values of the free drug percentage in the samples containing 120 µg/mL of
intralipid can also be attributed to the increased affinity of propofol to the lipid-modified HSA molecule. The whole collected
data set (see Figures 1_5) suggests also that the unbound propofol fraction changes tend to be far less pronounced at total
drug concentrations
concentrahigher than 3 µg/mL, that is, at the level which typically exists in the blood of patients when propofol total
anesthesia is maintained. Thus, the discussed effect is not likely to occur in total anesthesia conditions, provided that the
patients' hemodynamic parameters remain stable. Changes of hemodynamic parameters during anesthesia can cause free
propofol fraction change, as presented by Takizawa
et al[14].
The following conclusions were reached:
1. The presence of intralipid in protein samples does not suppress the rise of the unbound propofol fraction at small total
drug concentrations, however, the addition of intralipid noticeably lowers the free propofol percentage. The overall rise of
the free propofol fraction at a low total drug concentration, occurring even at the presence of intralipid, must not be ignored
when the drug is applied for sedative, antiemetic or other low-dosage purposes.
2. The relationship between the protein concentration and the unbound propofol fraction is almost linear.
3. The obtained results suggest that the protein samples spiked using ethanolic propofol solutions can be treated as
representative for human plasma samples taken from the patients receiving propofol infusions in the intralipid form.
4. The increase of the free propofol percentage induced by the decrease of the total drug concentration in the presence
of protein-binding intralipid suggests the need for a broader scope of research concerning drug-protein interactions,
particularly the influence of 1 drug total concentration on the free fraction of another drug. The present results are by no means
sufficient in this respect.
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