TOXICOLOGY
AND
APPLIED
Mechanism CHIU-WING
PHARMACOLOGY
of Transport LAM,? THEODGREJ.
*NASA
Biomedical
104,117-129
(1990)
and Distribution GALEN,~**JOHN
of Organic Solvents in Blood’ F. BOYD,-~ANDDUANEL.PIERSON*
Operations and Research Branch, and TKRUG Johnson Space Center, Houston, Texas 77058
Received
July 26. 1989: accepted
February
International,
21, 1990
Mechanism ofTransport and Distribution oforganic Solventsin Blood. LAM, C.-W., GALEN, J. F., AND PIERSON, D. L. (1990). Toxicol. Appl. Pharmacol. 104, 117-129. Little is known about the mechanism of transport and distribution of volatile organic compounds in blood. Studies were conducted on five typical organic solvents to investigate how these compounds are transported and distributed in blood. Groups of four to five rats were exposed for 2 hr to 500 ppm of n-hexane, toluene, chloroform, methyl isobutyl ketone (MIBK), or diethyl ether vapor; 94,66,90,5 1, or 49%, respectively, of these solvents in the blood were found in the red blood cells (RBCs). Very similar results were obtained in vitro when aqueous solutions of these solvents were added to rat blood. In vitro studies were also conducted on human blood with these solvents; 66, 43, 65, 49, or 46%, respectively, of the added solvent was taken up by the RBCs. These results indicate that RBCs from humans and rats exhibited substantial differences in affinity for the three more hydrophobic solvents studied. When solutions of these solvents were added to human plasma and RBC samples, large fractions (5 l-96%) of the solvents were recovered from ammonium sulfate-precipitated plasma proteins and hemoglobin. Smaller fractions were recovered from plasma water and red cell water. Less than 10% of each of the added solvents in RBC samples was found in the red cell membrane ghosts. These results indicate that RBCs play an important role in the uptake and transport of these solvents. Proteins, chiefly hemoglobin, are the major carriers of these compounds in blood. It can be inferred from the results of the present study that volatile lipophilic organic solvents are probably taken up by the hydrophobic sites of blood proteins. o 1990 Academic press, h. T. J., BOYD,
Little is known about the transport mechanism of volatile organic compounds (VOCs) in the blood. Of the VOCs, the general anesthetics have been best studied. The fact that the air:water partition coefficients (PCs) of many volatile anesthetics are very low has led to speculation that plasma water may not play a significant role in the transport of these compounds from the lung to the brain or other tissues. As pointed out by Franks and
Lieb ( 1982), “because of the low aqueous solubility, it is impossible, in principle, to deliver enough drug to the site, even at equilibrium.” Lam et al. ( 1986) also pointed out that transport of such hydrophobic organic compounds by plasma water to and from the brain would be slow and could not account for the rapid anesthetic induction and recovery. Many investigators have observed that volatile anesthetics dissolve to a greater extent in blood than in water or saline (Edelist et al., 1964; Eger and Larson, 1964; Larson et al., 1962; Robbins, 1936; Orcutt and Seevers, 1937). Recently, Gargas et al. (1989) found that the blood:air PCs are substantially larger than the saline:air PCs for 55 VOCs studied.
’ Portions of this work were presented at the 27th Annual Meeting of the Society of Toxicology. Dallas, TX, February 18,1988. * Present address: Lockheed Engineering and Sciences Co., 2400 NASA Road 1, C44. Houston, TX 77058.
117
004 1-008X/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
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LAM ET AL.
For example, the PCs for blood:air and for saline:air for n-hexane are 2.29 and 0.026, respectively; the corresponding coefficients for toluene are 18 and 1.75. Therefore, something in the blood other than the plasma water must take up the organic compounds. Early studies have been conducted to investigate the extent of interaction of volatile anesthetic agents with plasma, red blood cells (RBCs), red cell membranes, hemoglobin, and other blood proteins (Eger and Shargel, 1963; Grollman, 1929). However, these studies have not provided quantitative information on the contribution of these blood components to the transport of these agents. A common theory that anesthetics induce pharmacologic activity through membrane interaction (Franks and Lieb, 1982) coupled with the fact that organic compounds readily dissolve in oil or fat led to speculation that red blood cell membranes may transport a significant proportion of lipophilic agents in the blood. However, no data exist to support this speculation (Pang et al., 1980). Pang et al. followed up this speculation by assessing quantitatively the uptake of halothane by red cell membrane, hemoglobin, fatty acid-free albumin, and triglyceride using a dialysis system. Equations characterizing the halothane adsorption were derived. The results calculated using these equations indicated that membrane and blood water are the major carriers for halothane in blood. However, the relative distribution of halothane among different blood components varied greatly with blood halothane concentration (see Discussion). In a study of carbon disulfide (CS,), a volatile organic solvent that also possesses general anesthetic properties (Simpson, 1848), Lam et al. (1986) observed that about 90% of the inhaled CS2 in rats was transported by RBCs. It was proposed that RBCs may also play an important role in the transport of other organic solvents in blood. The present study was undertaken to further examine the role of RBCs in the transport of volatile organic solvents in blood. The distribution of organic
solvents in different blood components was also examined to reveal the mechanism of transport. Five organic solvents, namely, IIhexane, toluene, chloroform, methyl isobutyl ketone (MIBK), and diethyl ether, were chosen in order to cover a wide range of water solubility. The solubilities (w/w) of these compounds at ambient temperature are 0.001, 0.074, 0.815, 1.7, and 6.89%, respectively (Kreger, 1984). These widely used solvents are representatives of the aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ketones, and ethers. The present study also compared the blood partitioning of these organic solvents in in vivo and in vitro systems. Lam et al. (1986) showed that the partitioning of CS2 between RBCs and plasma of human blood in vitro was in good agreement with that observed in blood from rats exposed to CS2 by inhalation. The results from this study may provide some useful pharmacokinetic information for extrapolating data from rat to human. MATERIALS
AND
METHODS
Materials. Chloroform and glass-distilled methylene chloride were purchased from Burdick &Jackson Laboratories, Inc. (Muskegon. MI). MIBK and diethyl ether were supplied by Baker Chemical Co. (Phillipsburg, NJ). Toluene (99% mol pure) was obtained from Fischer Scientific (Houston, TX). These particular grades of methylene chloride and toluene were used because the impurities in these solvents did not interfere with chromatographic analyses in the present study. Human plasma was received from the Gulf Coast Blood Bank (Houston, TX). Human blood samples were generously provided by the NASA Clinical Laboratory (Houston, TX). Preparation
of aqueous
solutions
of organic
solvents.
Ten to 20 ml of n-hexane, toluene, chloroform, MIBK. or diethyl ether was added to 200 ml of isotonic saline buffered with 50 mM phosphate (pH 7.4). The mixtures, in capped glass bottles, were stirred vigorously overnight and then allowed to stand for at least 24 hr to allow the undissolved organic solvent to separate. The contents were chilled in an ice bath before aqueous aliquots were removed. Animals and animal care. Male Sprague-Dawley rata (HSD: (SD)BR) weighing about 300 g were obtained from Harlan Sprague-Dawley Farm (Houston, TX). The
SOLVENT
TRANSPORT
AND DISTRIBUTION
animals were housed in pairs in the vivarium at the Johnson Space Center on a 12-hr light-dark cycle and were provided with tap water and Purina Formulab Chow No. 5008 (Ralston Purina Co., St. Louis, MO). Animals were housed in the vivarium for at least 1 week before use. NIH guidelines for animal care were followed. Inhalation exposures. Groups of four to five rats were exposed to n-hexane, toluene, chloroform, MIBK, or diethyl ether in a 30-liter inhalation chamber (Leach, 1963). The vapor of the solvent under study was generated by bubbling nitrogen through the liquid solvent in an impinger, which was placed in a water bath (ambient temperature) or an ice bath (Lam and DiStefano, 1982). The concentrated vapor was mixed with house air (10 liters/min) in a 2-liter erlenmyer flask; the diluted vapor was then fed into the chamber. After the chamber atmosphere reached a steady-state concentration of approximately 500 ppm for at least 0.5 hr, rats were placed through a small door into the chamber one at a time, 10 min apart. After 2 hr of exposure, each rat was removed from the chamber and immediately decapitated for blood collection. Monitoring chamber vapor concentrations. Chamber vapor was drawn through Teflon tubing (i.d. 0.125 in.) into a gas injection loop/valve using a metal bellows pump (Metal Bellows Corp., Sharon, MA); the vapor stream was then circulated back to the chamber. During exposure, a sample was injected every 5-10 min into a gas chromatograph (GC) (HP 5730A, Hewlett Packard Co., Avondale, PA). The GC was equipped with a flame ionization detector and contained a 10-h stainless-steel column packed with 20% SP 2 100/O. 1% Carbowax 1500 (Supelco, Inc., Bellefonte, PA). The temperatures of the injector, oven, and detector were 150, 120, and 225°C respectively. The flow rates of helium, hydrogen, and air were 30,30, and 300 ml/min, respectively. The GC was calibrated as described below. Calibrations of the GCfor determining chamber vapor concentrations. A gas standard (100 ppm, uncertified) of MIBK purchased from Scatty Specialty Gas Co. (Houston, TX) did not give reproducible results when injected into the GC. The variation may have been partially due to the condensation of this high-boiling-point solvent in its container. Following a preliminary mass-balance study, a MIBK chamber concentration ofabout 500 ppm was generated. After the chamber concentration was stable for at least 0.5 hr, chamber MIBK vapor was drawn (approx 100 cc/ min) through two lo-ml, ice-cold methylene chloride traps and a flowmeter for 10 min using a peristaltic pump (Cole-Parmer, Chicago, IL). The chamber MIBK level was simultaneously monitored with a GC equipped with a gas-sample injection loop (HP 5730A). The concentration of MIBK in the methylene chloride was determined with another GC equipped with a liquid-sample injection port (HP 5830A) described in the following section.
IN BLOOD
119
MIBK standards in methylene chloride were prepared, and the amount of MIBK in the methylene chloride traps was calculated. During three vapor-trapping runs, most (98%) of the MIBK pulled through the methylene chloride traps was found to be retained in the first trap. The chamber concentrations, determined by this solventtrapping method, were 530,492, and 5 11 ppm. The average MIBK concentration with its corresponding average GC area signal from the HP 5730A was used as a singlepoint calibration for the GC (HP 5730A), which was then used for chamber vapor monitoring. Similar experiments were conducted with n-hexane using toluene as the trapping solvent. Essentially all (>99%) of the n-hexane vapor drawn through the traps was retained in the first trap. Three determinations were made and the chamber concentrations were found to be 452,453, and 432 ppm. Using an n-hexane gas standard of 100 ppm obtained from Scatty for the GC calibration, the chamber concentrations determined directly by GC during the three trapping runs were 460, 450, and 445 ppm, respectively. This trapping method was employed for toluene, chloroform, and diethyl ether GC calibrations. Determination of solvent partitioning between RBCs andplasma in bloodfrom exposed rats. After a 2-hr exposure to n-hexane, chloroform, toluene, MIBK, or diethyl ether, each rat was decapitated and blood was collected immediately in 2 ml of 3.8% ice-cold sodium citrate (pH 7.2) (Lam et al., 1986). After blood volume (normally 8-I I ml) was recorded, the sample was transferred to a chilled lo-ml glass tube (actual volume 13 ml) and capped. Samples were centrifuged for 10 min at 3000 rpm and the supematants (plasma plus citrate solution) were removed. Each RBC or plasma sample from rats exposed to n-hexane, chloroform, or diethyl ether was extracted with 2 or 5 ml oftoluene; the samples from rats exposed to toluene or MIBK were extracted with 5 ml of methylene chloride. After vigorous shaking, the concentrations of the n-hexane, chloroform, or diethyl ether in the toluene extracts and the toluene or MIBK in the methylene chloride extracts were determined using a GC (HP 5830A) equipped with a flame ionization detector and a column ofthe same type described above. The temperatures and gas flow rates were the same as those for the HP 5730A as detailed above. For all experiments, glassware was chilled prior to coming into contact with the biological samples. Samples were placed in an ice bath to minimize solvent loss. Small sample tubes with caps were chosen so as to minimize solvent loss into the head space. Determination of solvent partitioning between RBCs and plasma of rat blood in vitro. Four rats (350-450 g) were each anesthetized with 1 ml of Nembutal sodium solution (50 mg/ml, Abbott Laboratories, North Chicago, IL). Blood was drawn by cardiac puncture using 1Oml Venoject evacuated tubes (Terumo Medical, Elkton, MD) each containing a drop of K,EDTA. After the blood
120
LAM
samples were pooled and chilled, four IO-ml samples were placed in 13-ml capped glass tubes. One-half milliliter of an ice-cold isotonic buffer solution saturated with chloroform, toluene, MIBK. or diethyl ether or 2 ml of saturated n-hexane solution (the larger volume of n-hexane solution was used because its concentration in water was very low) was added to each blood sample. After gentle mixing, RBCs and plasma were separated. The contents of solvent present in the RBCs and plasma were determined as described above. Aliquots of 0.5 ml of the solvent solution (2 ml of n-hexane solution) were also added to 5-ml samples of phosphate buffer: the mixture was then extracted with methylene chloride or toluene to estimate the amount of solvent added to the blood samples. Since fresh rat blood samples were used in this experiment, and because the amounts of solvent in aqueous solution added to the blood were very small, hemolysis did not occur to any significant extent (by visual observation) and was not measured. Determination qf solvent partitioning between RBCs andplasma of human blood in vitro. Blood samples were collected from human volunteers with IO-ml Venoject tubes and used in experiments within 2 hr. The samples were pooled and chilled. Two to 4 ml of an ice-cold solution of n-hexane, chloroform, toluene, MIBK, or ether was added to 40 ml of chilled whole blood. In each experiment, eight 5-ml blood samples were dispensed. Four samples were extracted with methylene chloride or toluene to determine the initial amount of the added solvent in the blood samples. RBCs and plasma from the remaining four samples were separated and extracted with methylene chloride or toluene before the content of the added solvent was determined. Determination oj‘solvent distribution in plasma components in vitro. Ice-cold aqueous solutions (2-5 ml) of chloroform, toluene, MIBK, or diethyl ether ( IO ml of nhexane solution) were added to 40 ml of chilled human plasma. After mixing, eight 5-ml samples were pipetted into capped tubes. Four samples were assayedfor the initial concentration of the added solvent in the plasma. The other plasma samples were each treated with 5 g of ammonium sulfate dissolved in 5 ml of 10 miw phosphate buffer. The samples were then centrifuged at 20,OOOgfor 20 min. The supematants were removed and extracted with 2 or 5 ml ofmethylene chloride or toluene: the solvent concentration in the extracts was determined. Five milliliters of phosphate buffer was added to each precipitate in order to dissolve proteins before the methylene chloride or toluene extraction. The extracts were then assayed for the analyte. Determination cfsolvent distribution in RBCs in vitro. After removing plasma from chilled fresh human blood samples, 2 to 5 ml of an ice-cold solution of chloroform, toluene, MIBK, or diethyl ether or 20 ml of n-hexane solution was added to 20 ml of RBCs. After standing for 15 min in an ice bath, an equal volume of buffered iso-
ET AL. tonic saline was added to each sample for washing the RBCs. Sample were centrifuged, and the wash solution was removed. Eight 2-ml (2.15 g) RBC samples were placed in capped tubes. Four tubes were extracted with 5 ml of methylene chloride or toluene to assessthe concentration of added solvent in the sample. The other four RBC samples were each lysed with 10 ml phosphate buffer (10 mM, pH 7.4) and left standing in an ice bath for 30 min. The samples were then centrifuged for 40 min at 20,OOOg to precipitate the red cell membrane (Dodge et al., 1962). Each membrane fraction was extracted with 2 ml methylene chloride or toluene, and the solvent content in the membrane was determined. The hemolysates were each treated with 10 g ammonium sulfate (in 5 ml 10 mM phosphate) and centrifuged (20 min at 20,OOOg) to precipitate the hemoglobin. Supernatants were then removed. Hemoglobin and supernatant samples were extracted with methylene chloride or toluene before assaying for the quantity of analyte.
RESULTS Chamber Vapor Concentrations and Blood Levels of Volatile Organic Solvents Groups of four to five rats were each exposed for 2 hr to n-hexane, toluene, chloroform, MIBK, or diethyl ether at a target concentration of 500 ppm. The mean actual exposure concentrations were within f5% of the target concentration (Table I). The blood solvent concentrations, which are equal to the sum of the concentrations in the RBCs and plasma, are shown in Table 1. It should be pointed out that the momentary drop in chamber concentration each time an animal was removed for blood collection might have contributed to the varying of blood concentrations in the exposed animals. However, the variation did not affect the partitioning of the solvent between plasma and RBCs (Table 1). Distribution of Volatile Organic Solvents between Plasma and RBCs in the Blood of Exposed Rats Partitioning of organic solvents between plasma and RBCs was studied in the blood of exposed rats. Concentrations of these sol-
SOLVENT
TRANSPORT
AND DISTRIBUTION
121
IN BLOOD
TABLE 1 DISTRIBUTION OF WHEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER BETWEEN THE PLASMA AND RBCs OF INHALATION-EXPOSED RATS’ Solvent (mm) n-Hexane 515k25
Toluene 488 k 24
Chloroform 491 -t 39
MIBK 512+37
Ether’ 512k45
Rat (code) HI H2 H3 H4 Tl T2 T3 T4 T5 Cl c2 c3 c4 Ml M2 M3 M4 M5 El E2 E3
Plasma Wml)b 0.06 0.07 0.05 0.05 4.05 5.24 4.84 4.94 5.06 2.40 2.85 2.14 2.60 13.76 13.09 11.16 11.10 12.51 3.79 4.80 4.47
RBCs h/ml) b
Plasma + RBCs Wml)
1.09 0.86 0.77 0.73
RBCs x 100 RBCs + Plasma
Average
1.15 0.93 0.82 0.78 0.92
94.8 92.5 93.4 93.6 93.6
Average
10.90 15.03 14.98 15.02 15.09 14.20
62.8 65.1 67.7 67.1 66.4 65.9
Average
24.50 26.66 19.46 24.45 24.74
90.2 89.5 89.0 89.4 89.5
Average
27.73 27.49 22.50 23.60 25.18 25.30
50.4 52.4 50.0 52.9 50.3 51.2
Average
7.21 9.38 8.87 8.51
47.9 49.0 49.6 48.8
6.85 9.79 10.14 10.08 10.03 22.10 23.81 17.32 21.85 13.97 14.40 11.34 12.50 12.67 3.48 4.58 4.40
’ A group of four to five rats was exposed for 2 hr to the concentration (mean k SD) of the solvent indicated. h The concentration of solvent found by CC analysis in the plasma or RBCs from 1 ml of blood. ‘One blood sample was lost during preparation.
vents in plasma and RBCs were determined and are shown in Table 1. The average percentages of n-hexane, toluene, chloroform, MIBK, or diethyl ether in blood found in the RBCswere93.6,65.9, 89.5, 51.2,or48.8,respectively. The ratio of the amount of the solvent in RBCs to that in plasma was 14.7, 1.9, 8.5, 1.O, and 1.O, respectively. The results of this inhalation study show that n-hexane, toluene, and chloroform, the three more hydrophobic solvents, were taken up mainly by RBCs; MIBK and diethyl ether, which are
more water soluble, were carried in RBCs and plasma in approximately equal portions.
In Vitro Distribution of Volatile Organic Solvents between Plasma and RBCs in Rat Blood Partitioning of n-hexane, toluene, chloroform, MIBK, and diethyl ether between plasma and RBCs in vitro was also studied in order to compare the results with the in vivo
122
LAM
ET AL.
TABLE 2 DISTRIBUTIONOFI~-HEXANE,TOLLJENE,CHLOROFORM, MIBK, ANDDIETHYLETHERBETWEEN THEPLASMAAND RBCs OFRATBLOODSAMPLESTREATEDWITHTHESESOLVENTSIN VITRO”
Solvent n-Hexane Toluene Chloroform MIBK Ether
Plasma (mg/lO ml)’ 0.0019 0.056 0.46 3.68 10.78
f 0.0001 + 0.003 f 0.04 kO.08 + 0.16
RBCs (mg/lO ml)b 0.0229 0.130 2.34 3.82 9.67
f 0.00 I3 + 0.006 -co.15 + 0.06 + 0.34
RBCs x 100 RBCs + Plasma 92.4 69.9 83.8 50.9 47.3
a Four 0.5- or 2-ml aliquots of a saturated aqueous solution of each of these solvents were added to four lo-ml rat blood samples. The amount of added solvent partitioned into the plasma and RBCs was determined. Values are means + SD. b The amount of added solvent recovered from the plasma or RBCs of 10 ml of blood.
data obtained from exposed animals (Table 1). These in vitro results, shown in Table 2, agree well with those obtained from the blood of exposed rats (see comparison in Table 4). Again, n-hexane, chloroform, and toluene were found mainly in RBCs, while the distribution of MIBK and diethyl ether in RBCs and plasma was roughly equal. In this experiment, aliquots of each solvent in aqueous solution were also added to the buffer solutions in the same volumes as those added to the loml blood samples. The amount of n-hexane, toluene, chloroform, MIBK, or diethyl ether in the aliquots was 0.0239 +- 0.00 12, 0.172 + 0.032, 2.64 + 0.10, 7.29 +- 0.25, or 21.12 * 0.23 mg, respectively. If the recovery of each solvent from the buffer solution is taken as lOO%, then recovery of the added solvents from RBC and plasma samples was in the range of 96.8-l 10%.
In Vitro Distribution of Volatile Organic Solvents between Plasma and RBCs in Human Blood An in vitro study was also conducted in order to examine the distribution of n-hexane, toluene, chloroform, MIBK, and diethyl ether in human plasma and RBCs. The results are
shown in Table 3. The partitioning of n-hexane, toluene, or chloroform between the RBCs and plasma of human blood is substantially different from that of rat blood (Table 4); a sub stantially higher percentage of these hydrophobic solvents appeared in rat RBCs relative to human RBCs. In this experiment, the recovery of n-hexane, toluene, chloroform, MIBK, and diethyl ether in plasma and RBCs ranged from 90.3 to 108%, with the amount found in whole blood taken as 100% (Table 3).
Distribution of Volatile Organic Solvents in Human Plasma In order to investigate partitioning of solvents between plasma proteins and water in vitro, solvent-treated human plasma samples were precipitated with ammonium sulfate. The amount of solvent present in protein precipitates and in supernatants was determined and is shown in Table 5. A large fraction of each added solvent was found in the precipitates. The recovery of these solvents from plasma precipitates plus supernatants ranged from 82.6 to 99.1%, with the amount found in the plasma sample taken as 100% (Table 5).
SOLVENT
TRANSPORT
AND DISTRIBUTION
123
IN BLOOD
TABLE 3 DISTRIBUTION OF R-HEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER IN THE PLASMA AND RBCs OF HUMAN BLEND SAMPLES TREATED WITH THESE SOLVENTS IN VITRO’ Whole blood (w/5 ml)
Solvent n-Hexane Toluene Chloroform MIBK
0.0062
+ 0.0003
0.212 * 0.001 1.57 + 0.02 4.00
Ether
11.24
Plasma (w/5 ml) b 0.00 15 f 0.0003 0.126 + 0.006
RBCs (mg/S ml)b
RBCs x 100 Whole blood
0.0041 f 0.0003
65.6
0.092
f 0.005
0.53
kO.01
1.02
+ 0.02
42.1 64.7
to.12
2.34
k 0.08
1.98
f 0.06
49.4
k 0.53
6.15
to.11
5.15
kO.10
45.8
L?A saturated solution (2-5 ml) of each of these solvents was added to a 40-ml human blood sample. Four 5-ml aliquots (whole blood) of each sample were assayedfor the amount of added solvent. RBCs and plasma of the remaining four aliquots were separated before being assayed for solvent contents. Values are means + SD. ’ The amount of added solvent recovered from the plasma or RBCs from 5 ml of blood.
Distribution of Volatile Organic Solvents in Human RBCs In order to examine the distribution of these solvents in various red cell components, human RBCs were treated with each solvent in vitro. Table 6 shows the amounts of added solvent found in different RBC components. A large portion of each solvent added to RBCs was found in the hemoglobin fraction.
TABLE 4 COMPARISON OF THE IN VIVO AND IN VITRO SOLVENT PARTITIONING INTO RAT RBCs WITH THE IN VITRO SOLVENT PARTITIONING INTO HUMAN RBCs”
Solvent n-Hexane Toluene
Chloroform MIBK Ether
Rat, in vivob (RBCs/ Whole blood) x 100
Rat, in vitro’ (RBCs/ Whole blood) x loo
Human, in v&rod (RBCs/ Whole blood) x 100
93.6 65.9 89.5 51.2 48.8
92.4 69.9 83.8 50.9 47.3
65.6 42.1
64.7 49.4 45.8
’ The fraction of the organic solvent in the rat or human
blood
that
partitioned
into
The membrane ghosts accounted for less than 10% of the added solvents. However, it should be noted that the experiment was conducted using 2-ml samples of RBCs, in which cell water accounts for about 70% ( 1.4 ml) of the cell volume (Diem and Lentner, 1974a); a total of 15 ml of lysing and precipitating solutions was added to each RBC sample in order to fractionate membranes and hemoglobin from the aqueous phase. The addition of aqueous media would certainly remove a portion of the organic compound, especially the more hydrophilic solvents (MIBK and diethyl ether), from the hemoglobin and the membrane. Therefore, it might be expected that larger fractions of solvents in RBCs would be present in hemoglobin and membranes than are reflected by the values shown in Table 6. If the initial amount of solvent in the RBCs was taken as lOO%, then the recovery of chloroform, MIBK, and diethyl ether was 84. l100.1%; the recovery of n-hexane and toluene was about 75% (Table 6). DISCUSSION
the RBCs.
b Results from the last column of Table 1. ’ Results from the last column of Table 2. ’ Results from the last column of Table 3.
Studies were conducted with n-hexane, toluene, chloroform, MIBK, and diethyl ether
124
LAM ET AL. TABLE 5 DISTRIBUTION
Solvent n-Hexane Toluene Chloroform MIBK Ether
OF n-HEXANE. IN HUMAN PLASMA
Unit
TOLUENE, TREATED
CHLOROFORM, WITH THESE
MIBK, SOLVENTS
AND DIETHYL IN VITRO’
ETHER
Plasma
Plasma ppt
Plasma waterh
27.53 f 0.23 (100) 270.3 f 3.1 (100) 3.22 ? 0.01 (100) 4.54 t 0.06 (100) 12.81 kO.21 (100)
26.55 t 0.58 (96.4) 255.5 + 5.3 (94.6) 2.28 f 0.08 (70.8) 3.62 + 0.04 (79.7) 7.31 + 0.22 (57.1)
0. I7 t 0.06 (0.6) 12.5 t8.3 (4.6) 0.38 t 0.04 (11.7) 0.88 k 0.06 (19.5) 3.60 -+ 0.12 (28.1)
a A saturated solution of each of these solvents was added to 40 ml of human plasma sample. Four 5-ml aliquots of each sample were assayed for the amounts of added solvent and the values were set at 100%. Plasma from the remaining four aliquots was precipitated with ammonium sulfate. The solvent contents in supematants and precipitates were determined and the percentages were calculated using the values of column 3 as denominators. b Included plasma water and precipitating solutions.
to investigate the mechanism of volatile organic solvent transport in blood. The results in Table 4 show that RBCs are an important, perhaps the major, carrier for these solvents in blood. The solvents investigated in the present study differ in their hydrophobicity; therefore, it is expected that the partitioning profiles of these solvents between RBCs and plasma would differ. Following inhalation exposures of rats to these solvents individually, 94, 90, and 66% of blood n-hexane, chloroform, and toluene, respectively, were found in the RBCs. MIBK or diethyl ether were taken up in roughly equal proportions by RBCs and plasma (Table 1). The extent of distribution of these five solvents into RBCs is roughly parallel to the order of their hydrophobicity. An in vitro study of rat blood yielded similar results (Table 2). The similarity between in vivo and in vitro results from the same animal species (Table 4) is expected because the partitioning of these compounds in blood is based on their relative solubility in RBCs and plasma. Results obtained using this in vitro method with human blood could be useful in predicting the in vivo partitioning of an organic compound in human blood.
Results from in vitro studies of human blood show that the fractions of n-hexane, toluene, and chloroform that were partitioned into human RBCs were substantially less than the corresponding values in rat RBCs (Table 4). This greater uptake by rat RBCs has been observed by other investigators (Gingell, personal communication, 1989). Using a radiotracer technique, Gingell and his colleagues found that rat and human RBCs took up 75.2 and 36.6% of l,l-dibromo-2-chloropropane (DBCP) added in vitro to rat and human blood, respectively. The uptake percentages were independent of the DBCP concentration. These results support the current findings that rat RBCs have greater affinity for hydrophobic organic compounds than human RBCs. It is interesting to note that Gargas et al. (1989), using a vialequilibration technique, found that the rat bloodair PCs were substantially higher (the majority were 1S-2.0 times higher) than the corresponding human bloodair values for 32 of the 36 VOCs studied. In the present study, rat RBCs took up 94, 66, and 90% of the blood hexane, toluene, and chloroform, respectively; the corresponding values for hu-
SOLVENT
TRANSPORT
AND TABLE
DISTRIBUTION
125
IN BLOOD
6
DISTRIBUTION OF n-HEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER IN HUMAN RED BLOOD CELLS TREATED WITH THESE SOLVENTS IN VITRO“ Solvent
Unit
n-Hexane $) Toluene (2)
Chloroform ;; MIBK ;y
Ether g;
RBCs 18.56 k 2.66 (100) 145.3 k 7.7 (100) 1.21 20.02
(100) 2.11 k 0.05 (100) 3.33 2 0.25
(100)
Hemoglobin 13.36 + 0.66 (72.0) 101.7 k2.6 (70.0) 0.89 + 0.03 (73.6)
Membrane
Red cell waterb
0.99kO.11
Not detected
(5.3) 4.46 f 0.63 (3.1)
(
AND
APPLIED
Mechanism CHIU-WING
PHARMACOLOGY
of Transport LAM,? THEODGREJ.
*NASA
Biomedical
104,117-129
(1990)
and Distribution GALEN,~**JOHN
of Organic Solvents in Blood’ F. BOYD,-~ANDDUANEL.PIERSON*
Operations and Research Branch, and TKRUG Johnson Space Center, Houston, Texas 77058
Received
July 26. 1989: accepted
February
International,
21, 1990
Mechanism ofTransport and Distribution oforganic Solventsin Blood. LAM, C.-W., GALEN, J. F., AND PIERSON, D. L. (1990). Toxicol. Appl. Pharmacol. 104, 117-129. Little is known about the mechanism of transport and distribution of volatile organic compounds in blood. Studies were conducted on five typical organic solvents to investigate how these compounds are transported and distributed in blood. Groups of four to five rats were exposed for 2 hr to 500 ppm of n-hexane, toluene, chloroform, methyl isobutyl ketone (MIBK), or diethyl ether vapor; 94,66,90,5 1, or 49%, respectively, of these solvents in the blood were found in the red blood cells (RBCs). Very similar results were obtained in vitro when aqueous solutions of these solvents were added to rat blood. In vitro studies were also conducted on human blood with these solvents; 66, 43, 65, 49, or 46%, respectively, of the added solvent was taken up by the RBCs. These results indicate that RBCs from humans and rats exhibited substantial differences in affinity for the three more hydrophobic solvents studied. When solutions of these solvents were added to human plasma and RBC samples, large fractions (5 l-96%) of the solvents were recovered from ammonium sulfate-precipitated plasma proteins and hemoglobin. Smaller fractions were recovered from plasma water and red cell water. Less than 10% of each of the added solvents in RBC samples was found in the red cell membrane ghosts. These results indicate that RBCs play an important role in the uptake and transport of these solvents. Proteins, chiefly hemoglobin, are the major carriers of these compounds in blood. It can be inferred from the results of the present study that volatile lipophilic organic solvents are probably taken up by the hydrophobic sites of blood proteins. o 1990 Academic press, h. T. J., BOYD,
Little is known about the transport mechanism of volatile organic compounds (VOCs) in the blood. Of the VOCs, the general anesthetics have been best studied. The fact that the air:water partition coefficients (PCs) of many volatile anesthetics are very low has led to speculation that plasma water may not play a significant role in the transport of these compounds from the lung to the brain or other tissues. As pointed out by Franks and
Lieb ( 1982), “because of the low aqueous solubility, it is impossible, in principle, to deliver enough drug to the site, even at equilibrium.” Lam et al. ( 1986) also pointed out that transport of such hydrophobic organic compounds by plasma water to and from the brain would be slow and could not account for the rapid anesthetic induction and recovery. Many investigators have observed that volatile anesthetics dissolve to a greater extent in blood than in water or saline (Edelist et al., 1964; Eger and Larson, 1964; Larson et al., 1962; Robbins, 1936; Orcutt and Seevers, 1937). Recently, Gargas et al. (1989) found that the blood:air PCs are substantially larger than the saline:air PCs for 55 VOCs studied.
’ Portions of this work were presented at the 27th Annual Meeting of the Society of Toxicology. Dallas, TX, February 18,1988. * Present address: Lockheed Engineering and Sciences Co., 2400 NASA Road 1, C44. Houston, TX 77058.
117
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$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
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LAM ET AL.
For example, the PCs for blood:air and for saline:air for n-hexane are 2.29 and 0.026, respectively; the corresponding coefficients for toluene are 18 and 1.75. Therefore, something in the blood other than the plasma water must take up the organic compounds. Early studies have been conducted to investigate the extent of interaction of volatile anesthetic agents with plasma, red blood cells (RBCs), red cell membranes, hemoglobin, and other blood proteins (Eger and Shargel, 1963; Grollman, 1929). However, these studies have not provided quantitative information on the contribution of these blood components to the transport of these agents. A common theory that anesthetics induce pharmacologic activity through membrane interaction (Franks and Lieb, 1982) coupled with the fact that organic compounds readily dissolve in oil or fat led to speculation that red blood cell membranes may transport a significant proportion of lipophilic agents in the blood. However, no data exist to support this speculation (Pang et al., 1980). Pang et al. followed up this speculation by assessing quantitatively the uptake of halothane by red cell membrane, hemoglobin, fatty acid-free albumin, and triglyceride using a dialysis system. Equations characterizing the halothane adsorption were derived. The results calculated using these equations indicated that membrane and blood water are the major carriers for halothane in blood. However, the relative distribution of halothane among different blood components varied greatly with blood halothane concentration (see Discussion). In a study of carbon disulfide (CS,), a volatile organic solvent that also possesses general anesthetic properties (Simpson, 1848), Lam et al. (1986) observed that about 90% of the inhaled CS2 in rats was transported by RBCs. It was proposed that RBCs may also play an important role in the transport of other organic solvents in blood. The present study was undertaken to further examine the role of RBCs in the transport of volatile organic solvents in blood. The distribution of organic
solvents in different blood components was also examined to reveal the mechanism of transport. Five organic solvents, namely, IIhexane, toluene, chloroform, methyl isobutyl ketone (MIBK), and diethyl ether, were chosen in order to cover a wide range of water solubility. The solubilities (w/w) of these compounds at ambient temperature are 0.001, 0.074, 0.815, 1.7, and 6.89%, respectively (Kreger, 1984). These widely used solvents are representatives of the aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ketones, and ethers. The present study also compared the blood partitioning of these organic solvents in in vivo and in vitro systems. Lam et al. (1986) showed that the partitioning of CS2 between RBCs and plasma of human blood in vitro was in good agreement with that observed in blood from rats exposed to CS2 by inhalation. The results from this study may provide some useful pharmacokinetic information for extrapolating data from rat to human. MATERIALS
AND
METHODS
Materials. Chloroform and glass-distilled methylene chloride were purchased from Burdick &Jackson Laboratories, Inc. (Muskegon. MI). MIBK and diethyl ether were supplied by Baker Chemical Co. (Phillipsburg, NJ). Toluene (99% mol pure) was obtained from Fischer Scientific (Houston, TX). These particular grades of methylene chloride and toluene were used because the impurities in these solvents did not interfere with chromatographic analyses in the present study. Human plasma was received from the Gulf Coast Blood Bank (Houston, TX). Human blood samples were generously provided by the NASA Clinical Laboratory (Houston, TX). Preparation
of aqueous
solutions
of organic
solvents.
Ten to 20 ml of n-hexane, toluene, chloroform, MIBK. or diethyl ether was added to 200 ml of isotonic saline buffered with 50 mM phosphate (pH 7.4). The mixtures, in capped glass bottles, were stirred vigorously overnight and then allowed to stand for at least 24 hr to allow the undissolved organic solvent to separate. The contents were chilled in an ice bath before aqueous aliquots were removed. Animals and animal care. Male Sprague-Dawley rata (HSD: (SD)BR) weighing about 300 g were obtained from Harlan Sprague-Dawley Farm (Houston, TX). The
SOLVENT
TRANSPORT
AND DISTRIBUTION
animals were housed in pairs in the vivarium at the Johnson Space Center on a 12-hr light-dark cycle and were provided with tap water and Purina Formulab Chow No. 5008 (Ralston Purina Co., St. Louis, MO). Animals were housed in the vivarium for at least 1 week before use. NIH guidelines for animal care were followed. Inhalation exposures. Groups of four to five rats were exposed to n-hexane, toluene, chloroform, MIBK, or diethyl ether in a 30-liter inhalation chamber (Leach, 1963). The vapor of the solvent under study was generated by bubbling nitrogen through the liquid solvent in an impinger, which was placed in a water bath (ambient temperature) or an ice bath (Lam and DiStefano, 1982). The concentrated vapor was mixed with house air (10 liters/min) in a 2-liter erlenmyer flask; the diluted vapor was then fed into the chamber. After the chamber atmosphere reached a steady-state concentration of approximately 500 ppm for at least 0.5 hr, rats were placed through a small door into the chamber one at a time, 10 min apart. After 2 hr of exposure, each rat was removed from the chamber and immediately decapitated for blood collection. Monitoring chamber vapor concentrations. Chamber vapor was drawn through Teflon tubing (i.d. 0.125 in.) into a gas injection loop/valve using a metal bellows pump (Metal Bellows Corp., Sharon, MA); the vapor stream was then circulated back to the chamber. During exposure, a sample was injected every 5-10 min into a gas chromatograph (GC) (HP 5730A, Hewlett Packard Co., Avondale, PA). The GC was equipped with a flame ionization detector and contained a 10-h stainless-steel column packed with 20% SP 2 100/O. 1% Carbowax 1500 (Supelco, Inc., Bellefonte, PA). The temperatures of the injector, oven, and detector were 150, 120, and 225°C respectively. The flow rates of helium, hydrogen, and air were 30,30, and 300 ml/min, respectively. The GC was calibrated as described below. Calibrations of the GCfor determining chamber vapor concentrations. A gas standard (100 ppm, uncertified) of MIBK purchased from Scatty Specialty Gas Co. (Houston, TX) did not give reproducible results when injected into the GC. The variation may have been partially due to the condensation of this high-boiling-point solvent in its container. Following a preliminary mass-balance study, a MIBK chamber concentration ofabout 500 ppm was generated. After the chamber concentration was stable for at least 0.5 hr, chamber MIBK vapor was drawn (approx 100 cc/ min) through two lo-ml, ice-cold methylene chloride traps and a flowmeter for 10 min using a peristaltic pump (Cole-Parmer, Chicago, IL). The chamber MIBK level was simultaneously monitored with a GC equipped with a gas-sample injection loop (HP 5730A). The concentration of MIBK in the methylene chloride was determined with another GC equipped with a liquid-sample injection port (HP 5830A) described in the following section.
IN BLOOD
119
MIBK standards in methylene chloride were prepared, and the amount of MIBK in the methylene chloride traps was calculated. During three vapor-trapping runs, most (98%) of the MIBK pulled through the methylene chloride traps was found to be retained in the first trap. The chamber concentrations, determined by this solventtrapping method, were 530,492, and 5 11 ppm. The average MIBK concentration with its corresponding average GC area signal from the HP 5730A was used as a singlepoint calibration for the GC (HP 5730A), which was then used for chamber vapor monitoring. Similar experiments were conducted with n-hexane using toluene as the trapping solvent. Essentially all (>99%) of the n-hexane vapor drawn through the traps was retained in the first trap. Three determinations were made and the chamber concentrations were found to be 452,453, and 432 ppm. Using an n-hexane gas standard of 100 ppm obtained from Scatty for the GC calibration, the chamber concentrations determined directly by GC during the three trapping runs were 460, 450, and 445 ppm, respectively. This trapping method was employed for toluene, chloroform, and diethyl ether GC calibrations. Determination of solvent partitioning between RBCs andplasma in bloodfrom exposed rats. After a 2-hr exposure to n-hexane, chloroform, toluene, MIBK, or diethyl ether, each rat was decapitated and blood was collected immediately in 2 ml of 3.8% ice-cold sodium citrate (pH 7.2) (Lam et al., 1986). After blood volume (normally 8-I I ml) was recorded, the sample was transferred to a chilled lo-ml glass tube (actual volume 13 ml) and capped. Samples were centrifuged for 10 min at 3000 rpm and the supematants (plasma plus citrate solution) were removed. Each RBC or plasma sample from rats exposed to n-hexane, chloroform, or diethyl ether was extracted with 2 or 5 ml oftoluene; the samples from rats exposed to toluene or MIBK were extracted with 5 ml of methylene chloride. After vigorous shaking, the concentrations of the n-hexane, chloroform, or diethyl ether in the toluene extracts and the toluene or MIBK in the methylene chloride extracts were determined using a GC (HP 5830A) equipped with a flame ionization detector and a column ofthe same type described above. The temperatures and gas flow rates were the same as those for the HP 5730A as detailed above. For all experiments, glassware was chilled prior to coming into contact with the biological samples. Samples were placed in an ice bath to minimize solvent loss. Small sample tubes with caps were chosen so as to minimize solvent loss into the head space. Determination of solvent partitioning between RBCs and plasma of rat blood in vitro. Four rats (350-450 g) were each anesthetized with 1 ml of Nembutal sodium solution (50 mg/ml, Abbott Laboratories, North Chicago, IL). Blood was drawn by cardiac puncture using 1Oml Venoject evacuated tubes (Terumo Medical, Elkton, MD) each containing a drop of K,EDTA. After the blood
120
LAM
samples were pooled and chilled, four IO-ml samples were placed in 13-ml capped glass tubes. One-half milliliter of an ice-cold isotonic buffer solution saturated with chloroform, toluene, MIBK. or diethyl ether or 2 ml of saturated n-hexane solution (the larger volume of n-hexane solution was used because its concentration in water was very low) was added to each blood sample. After gentle mixing, RBCs and plasma were separated. The contents of solvent present in the RBCs and plasma were determined as described above. Aliquots of 0.5 ml of the solvent solution (2 ml of n-hexane solution) were also added to 5-ml samples of phosphate buffer: the mixture was then extracted with methylene chloride or toluene to estimate the amount of solvent added to the blood samples. Since fresh rat blood samples were used in this experiment, and because the amounts of solvent in aqueous solution added to the blood were very small, hemolysis did not occur to any significant extent (by visual observation) and was not measured. Determination qf solvent partitioning between RBCs andplasma of human blood in vitro. Blood samples were collected from human volunteers with IO-ml Venoject tubes and used in experiments within 2 hr. The samples were pooled and chilled. Two to 4 ml of an ice-cold solution of n-hexane, chloroform, toluene, MIBK, or ether was added to 40 ml of chilled whole blood. In each experiment, eight 5-ml blood samples were dispensed. Four samples were extracted with methylene chloride or toluene to determine the initial amount of the added solvent in the blood samples. RBCs and plasma from the remaining four samples were separated and extracted with methylene chloride or toluene before the content of the added solvent was determined. Determination oj‘solvent distribution in plasma components in vitro. Ice-cold aqueous solutions (2-5 ml) of chloroform, toluene, MIBK, or diethyl ether ( IO ml of nhexane solution) were added to 40 ml of chilled human plasma. After mixing, eight 5-ml samples were pipetted into capped tubes. Four samples were assayedfor the initial concentration of the added solvent in the plasma. The other plasma samples were each treated with 5 g of ammonium sulfate dissolved in 5 ml of 10 miw phosphate buffer. The samples were then centrifuged at 20,OOOgfor 20 min. The supematants were removed and extracted with 2 or 5 ml ofmethylene chloride or toluene: the solvent concentration in the extracts was determined. Five milliliters of phosphate buffer was added to each precipitate in order to dissolve proteins before the methylene chloride or toluene extraction. The extracts were then assayed for the analyte. Determination cfsolvent distribution in RBCs in vitro. After removing plasma from chilled fresh human blood samples, 2 to 5 ml of an ice-cold solution of chloroform, toluene, MIBK, or diethyl ether or 20 ml of n-hexane solution was added to 20 ml of RBCs. After standing for 15 min in an ice bath, an equal volume of buffered iso-
ET AL. tonic saline was added to each sample for washing the RBCs. Sample were centrifuged, and the wash solution was removed. Eight 2-ml (2.15 g) RBC samples were placed in capped tubes. Four tubes were extracted with 5 ml of methylene chloride or toluene to assessthe concentration of added solvent in the sample. The other four RBC samples were each lysed with 10 ml phosphate buffer (10 mM, pH 7.4) and left standing in an ice bath for 30 min. The samples were then centrifuged for 40 min at 20,OOOg to precipitate the red cell membrane (Dodge et al., 1962). Each membrane fraction was extracted with 2 ml methylene chloride or toluene, and the solvent content in the membrane was determined. The hemolysates were each treated with 10 g ammonium sulfate (in 5 ml 10 mM phosphate) and centrifuged (20 min at 20,OOOg) to precipitate the hemoglobin. Supernatants were then removed. Hemoglobin and supernatant samples were extracted with methylene chloride or toluene before assaying for the quantity of analyte.
RESULTS Chamber Vapor Concentrations and Blood Levels of Volatile Organic Solvents Groups of four to five rats were each exposed for 2 hr to n-hexane, toluene, chloroform, MIBK, or diethyl ether at a target concentration of 500 ppm. The mean actual exposure concentrations were within f5% of the target concentration (Table I). The blood solvent concentrations, which are equal to the sum of the concentrations in the RBCs and plasma, are shown in Table 1. It should be pointed out that the momentary drop in chamber concentration each time an animal was removed for blood collection might have contributed to the varying of blood concentrations in the exposed animals. However, the variation did not affect the partitioning of the solvent between plasma and RBCs (Table 1). Distribution of Volatile Organic Solvents between Plasma and RBCs in the Blood of Exposed Rats Partitioning of organic solvents between plasma and RBCs was studied in the blood of exposed rats. Concentrations of these sol-
SOLVENT
TRANSPORT
AND DISTRIBUTION
121
IN BLOOD
TABLE 1 DISTRIBUTION OF WHEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER BETWEEN THE PLASMA AND RBCs OF INHALATION-EXPOSED RATS’ Solvent (mm) n-Hexane 515k25
Toluene 488 k 24
Chloroform 491 -t 39
MIBK 512+37
Ether’ 512k45
Rat (code) HI H2 H3 H4 Tl T2 T3 T4 T5 Cl c2 c3 c4 Ml M2 M3 M4 M5 El E2 E3
Plasma Wml)b 0.06 0.07 0.05 0.05 4.05 5.24 4.84 4.94 5.06 2.40 2.85 2.14 2.60 13.76 13.09 11.16 11.10 12.51 3.79 4.80 4.47
RBCs h/ml) b
Plasma + RBCs Wml)
1.09 0.86 0.77 0.73
RBCs x 100 RBCs + Plasma
Average
1.15 0.93 0.82 0.78 0.92
94.8 92.5 93.4 93.6 93.6
Average
10.90 15.03 14.98 15.02 15.09 14.20
62.8 65.1 67.7 67.1 66.4 65.9
Average
24.50 26.66 19.46 24.45 24.74
90.2 89.5 89.0 89.4 89.5
Average
27.73 27.49 22.50 23.60 25.18 25.30
50.4 52.4 50.0 52.9 50.3 51.2
Average
7.21 9.38 8.87 8.51
47.9 49.0 49.6 48.8
6.85 9.79 10.14 10.08 10.03 22.10 23.81 17.32 21.85 13.97 14.40 11.34 12.50 12.67 3.48 4.58 4.40
’ A group of four to five rats was exposed for 2 hr to the concentration (mean k SD) of the solvent indicated. h The concentration of solvent found by CC analysis in the plasma or RBCs from 1 ml of blood. ‘One blood sample was lost during preparation.
vents in plasma and RBCs were determined and are shown in Table 1. The average percentages of n-hexane, toluene, chloroform, MIBK, or diethyl ether in blood found in the RBCswere93.6,65.9, 89.5, 51.2,or48.8,respectively. The ratio of the amount of the solvent in RBCs to that in plasma was 14.7, 1.9, 8.5, 1.O, and 1.O, respectively. The results of this inhalation study show that n-hexane, toluene, and chloroform, the three more hydrophobic solvents, were taken up mainly by RBCs; MIBK and diethyl ether, which are
more water soluble, were carried in RBCs and plasma in approximately equal portions.
In Vitro Distribution of Volatile Organic Solvents between Plasma and RBCs in Rat Blood Partitioning of n-hexane, toluene, chloroform, MIBK, and diethyl ether between plasma and RBCs in vitro was also studied in order to compare the results with the in vivo
122
LAM
ET AL.
TABLE 2 DISTRIBUTIONOFI~-HEXANE,TOLLJENE,CHLOROFORM, MIBK, ANDDIETHYLETHERBETWEEN THEPLASMAAND RBCs OFRATBLOODSAMPLESTREATEDWITHTHESESOLVENTSIN VITRO”
Solvent n-Hexane Toluene Chloroform MIBK Ether
Plasma (mg/lO ml)’ 0.0019 0.056 0.46 3.68 10.78
f 0.0001 + 0.003 f 0.04 kO.08 + 0.16
RBCs (mg/lO ml)b 0.0229 0.130 2.34 3.82 9.67
f 0.00 I3 + 0.006 -co.15 + 0.06 + 0.34
RBCs x 100 RBCs + Plasma 92.4 69.9 83.8 50.9 47.3
a Four 0.5- or 2-ml aliquots of a saturated aqueous solution of each of these solvents were added to four lo-ml rat blood samples. The amount of added solvent partitioned into the plasma and RBCs was determined. Values are means + SD. b The amount of added solvent recovered from the plasma or RBCs of 10 ml of blood.
data obtained from exposed animals (Table 1). These in vitro results, shown in Table 2, agree well with those obtained from the blood of exposed rats (see comparison in Table 4). Again, n-hexane, chloroform, and toluene were found mainly in RBCs, while the distribution of MIBK and diethyl ether in RBCs and plasma was roughly equal. In this experiment, aliquots of each solvent in aqueous solution were also added to the buffer solutions in the same volumes as those added to the loml blood samples. The amount of n-hexane, toluene, chloroform, MIBK, or diethyl ether in the aliquots was 0.0239 +- 0.00 12, 0.172 + 0.032, 2.64 + 0.10, 7.29 +- 0.25, or 21.12 * 0.23 mg, respectively. If the recovery of each solvent from the buffer solution is taken as lOO%, then recovery of the added solvents from RBC and plasma samples was in the range of 96.8-l 10%.
In Vitro Distribution of Volatile Organic Solvents between Plasma and RBCs in Human Blood An in vitro study was also conducted in order to examine the distribution of n-hexane, toluene, chloroform, MIBK, and diethyl ether in human plasma and RBCs. The results are
shown in Table 3. The partitioning of n-hexane, toluene, or chloroform between the RBCs and plasma of human blood is substantially different from that of rat blood (Table 4); a sub stantially higher percentage of these hydrophobic solvents appeared in rat RBCs relative to human RBCs. In this experiment, the recovery of n-hexane, toluene, chloroform, MIBK, and diethyl ether in plasma and RBCs ranged from 90.3 to 108%, with the amount found in whole blood taken as 100% (Table 3).
Distribution of Volatile Organic Solvents in Human Plasma In order to investigate partitioning of solvents between plasma proteins and water in vitro, solvent-treated human plasma samples were precipitated with ammonium sulfate. The amount of solvent present in protein precipitates and in supernatants was determined and is shown in Table 5. A large fraction of each added solvent was found in the precipitates. The recovery of these solvents from plasma precipitates plus supernatants ranged from 82.6 to 99.1%, with the amount found in the plasma sample taken as 100% (Table 5).
SOLVENT
TRANSPORT
AND DISTRIBUTION
123
IN BLOOD
TABLE 3 DISTRIBUTION OF R-HEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER IN THE PLASMA AND RBCs OF HUMAN BLEND SAMPLES TREATED WITH THESE SOLVENTS IN VITRO’ Whole blood (w/5 ml)
Solvent n-Hexane Toluene Chloroform MIBK
0.0062
+ 0.0003
0.212 * 0.001 1.57 + 0.02 4.00
Ether
11.24
Plasma (w/5 ml) b 0.00 15 f 0.0003 0.126 + 0.006
RBCs (mg/S ml)b
RBCs x 100 Whole blood
0.0041 f 0.0003
65.6
0.092
f 0.005
0.53
kO.01
1.02
+ 0.02
42.1 64.7
to.12
2.34
k 0.08
1.98
f 0.06
49.4
k 0.53
6.15
to.11
5.15
kO.10
45.8
L?A saturated solution (2-5 ml) of each of these solvents was added to a 40-ml human blood sample. Four 5-ml aliquots (whole blood) of each sample were assayedfor the amount of added solvent. RBCs and plasma of the remaining four aliquots were separated before being assayed for solvent contents. Values are means + SD. ’ The amount of added solvent recovered from the plasma or RBCs from 5 ml of blood.
Distribution of Volatile Organic Solvents in Human RBCs In order to examine the distribution of these solvents in various red cell components, human RBCs were treated with each solvent in vitro. Table 6 shows the amounts of added solvent found in different RBC components. A large portion of each solvent added to RBCs was found in the hemoglobin fraction.
TABLE 4 COMPARISON OF THE IN VIVO AND IN VITRO SOLVENT PARTITIONING INTO RAT RBCs WITH THE IN VITRO SOLVENT PARTITIONING INTO HUMAN RBCs”
Solvent n-Hexane Toluene
Chloroform MIBK Ether
Rat, in vivob (RBCs/ Whole blood) x 100
Rat, in vitro’ (RBCs/ Whole blood) x loo
Human, in v&rod (RBCs/ Whole blood) x 100
93.6 65.9 89.5 51.2 48.8
92.4 69.9 83.8 50.9 47.3
65.6 42.1
64.7 49.4 45.8
’ The fraction of the organic solvent in the rat or human
blood
that
partitioned
into
The membrane ghosts accounted for less than 10% of the added solvents. However, it should be noted that the experiment was conducted using 2-ml samples of RBCs, in which cell water accounts for about 70% ( 1.4 ml) of the cell volume (Diem and Lentner, 1974a); a total of 15 ml of lysing and precipitating solutions was added to each RBC sample in order to fractionate membranes and hemoglobin from the aqueous phase. The addition of aqueous media would certainly remove a portion of the organic compound, especially the more hydrophilic solvents (MIBK and diethyl ether), from the hemoglobin and the membrane. Therefore, it might be expected that larger fractions of solvents in RBCs would be present in hemoglobin and membranes than are reflected by the values shown in Table 6. If the initial amount of solvent in the RBCs was taken as lOO%, then the recovery of chloroform, MIBK, and diethyl ether was 84. l100.1%; the recovery of n-hexane and toluene was about 75% (Table 6). DISCUSSION
the RBCs.
b Results from the last column of Table 1. ’ Results from the last column of Table 2. ’ Results from the last column of Table 3.
Studies were conducted with n-hexane, toluene, chloroform, MIBK, and diethyl ether
124
LAM ET AL. TABLE 5 DISTRIBUTION
Solvent n-Hexane Toluene Chloroform MIBK Ether
OF n-HEXANE. IN HUMAN PLASMA
Unit
TOLUENE, TREATED
CHLOROFORM, WITH THESE
MIBK, SOLVENTS
AND DIETHYL IN VITRO’
ETHER
Plasma
Plasma ppt
Plasma waterh
27.53 f 0.23 (100) 270.3 f 3.1 (100) 3.22 ? 0.01 (100) 4.54 t 0.06 (100) 12.81 kO.21 (100)
26.55 t 0.58 (96.4) 255.5 + 5.3 (94.6) 2.28 f 0.08 (70.8) 3.62 + 0.04 (79.7) 7.31 + 0.22 (57.1)
0. I7 t 0.06 (0.6) 12.5 t8.3 (4.6) 0.38 t 0.04 (11.7) 0.88 k 0.06 (19.5) 3.60 -+ 0.12 (28.1)
a A saturated solution of each of these solvents was added to 40 ml of human plasma sample. Four 5-ml aliquots of each sample were assayed for the amounts of added solvent and the values were set at 100%. Plasma from the remaining four aliquots was precipitated with ammonium sulfate. The solvent contents in supematants and precipitates were determined and the percentages were calculated using the values of column 3 as denominators. b Included plasma water and precipitating solutions.
to investigate the mechanism of volatile organic solvent transport in blood. The results in Table 4 show that RBCs are an important, perhaps the major, carrier for these solvents in blood. The solvents investigated in the present study differ in their hydrophobicity; therefore, it is expected that the partitioning profiles of these solvents between RBCs and plasma would differ. Following inhalation exposures of rats to these solvents individually, 94, 90, and 66% of blood n-hexane, chloroform, and toluene, respectively, were found in the RBCs. MIBK or diethyl ether were taken up in roughly equal proportions by RBCs and plasma (Table 1). The extent of distribution of these five solvents into RBCs is roughly parallel to the order of their hydrophobicity. An in vitro study of rat blood yielded similar results (Table 2). The similarity between in vivo and in vitro results from the same animal species (Table 4) is expected because the partitioning of these compounds in blood is based on their relative solubility in RBCs and plasma. Results obtained using this in vitro method with human blood could be useful in predicting the in vivo partitioning of an organic compound in human blood.
Results from in vitro studies of human blood show that the fractions of n-hexane, toluene, and chloroform that were partitioned into human RBCs were substantially less than the corresponding values in rat RBCs (Table 4). This greater uptake by rat RBCs has been observed by other investigators (Gingell, personal communication, 1989). Using a radiotracer technique, Gingell and his colleagues found that rat and human RBCs took up 75.2 and 36.6% of l,l-dibromo-2-chloropropane (DBCP) added in vitro to rat and human blood, respectively. The uptake percentages were independent of the DBCP concentration. These results support the current findings that rat RBCs have greater affinity for hydrophobic organic compounds than human RBCs. It is interesting to note that Gargas et al. (1989), using a vialequilibration technique, found that the rat bloodair PCs were substantially higher (the majority were 1S-2.0 times higher) than the corresponding human bloodair values for 32 of the 36 VOCs studied. In the present study, rat RBCs took up 94, 66, and 90% of the blood hexane, toluene, and chloroform, respectively; the corresponding values for hu-
SOLVENT
TRANSPORT
AND TABLE
DISTRIBUTION
125
IN BLOOD
6
DISTRIBUTION OF n-HEXANE, TOLUENE, CHLOROFORM, MIBK, AND DIETHYL ETHER IN HUMAN RED BLOOD CELLS TREATED WITH THESE SOLVENTS IN VITRO“ Solvent
Unit
n-Hexane $) Toluene (2)
Chloroform ;; MIBK ;y
Ether g;
RBCs 18.56 k 2.66 (100) 145.3 k 7.7 (100) 1.21 20.02
(100) 2.11 k 0.05 (100) 3.33 2 0.25
(100)
Hemoglobin 13.36 + 0.66 (72.0) 101.7 k2.6 (70.0) 0.89 + 0.03 (73.6)
Membrane
Red cell waterb
0.99kO.11
Not detected
(5.3) 4.46 f 0.63 (3.1)
(
