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Studies on the fate of fat emulsions following intravenous administration to rabbits and the effect of added electrolyte
CLINICAL
NUTRITION
(1987) 6: 13-19
Studies on the Fate ofFat Emulsions Following Intravenous Administration to Rabbits and thk Effect of Added Electrolyte S. S. Davis*, L. Illumt,
P. West* and M. Galloway*
*Department of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK. tDepartment of Pharmaceutics, Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, (Reprint requests to S.S.D.)
Denmark.
The effect of the fat emulsion Intralipid$+ (soybean oil emulsified with egg lecithin) on ABSTRACT reticuloendothelial function has been evaluated in rabbits using the non-invasive technique of gamma scintigraphy. Neither acute nor chronic administration of the fat emulsion had any significant effect on the subsequent uptake of labelled test colloids (microspheres and fat emulsions) into the liver and spleen. Studies were also made on the uptake of labelled fat emulsions that had modified physical properties as caused by the addition of electrolyte (calcium chloride). Flocculated emulsions and those carrying a positive charge (charge reversed) demonstrated different patterns of uptake in the liver/spleen and heart/lung regions of the rabbit immediately after intravenous administration. The results are discussed in terms of entrappment of emulsion Aoccules in the capillary beds of the lungs and biological
fate of positively
charged
colloidal
particles.
Mg+ + greater than about 10 mmol 1 -I) the fat particles can even carry a net positive charge (+ 15 mV) [‘7]. The effects of these physical changes on the in vivo behaviour of fat emulsions had nof been studied in detail. Apparently it is assumed that any effect will be reversed easily upon dilution of the mixed emulsion when injected into the blood. This assumption may not be valid since the adsorption of blood components onto emulsion droplets occurs very rapidly [8] and therefore the possibility exists of an enhanced state of flocculation of droplets in vivo or an altered adsorption of blood components (e.g. type of apoprotein). Therefore, we have conducted a series of studies using the non-invasive technique of gamma scintigraphy to follow the whole body deposition of fat emulsions and thereby to evaluate the effect on reticuloendothelial function. The rabbit has been used as the animal model.
INTRODUCTION Emulsions prepared from vegetable oils such as soybean oil (and more recently sesame oil) emulsified with egg or soy phosphatides, have been used with success in parenteral nutrition with remarkably few adverse reactions and side effects 111. Their fate in the body, as determined by the adsorption of apolipoproteins and subsequent clearance from the blood, has been found similar to that of the endogenous lipid particles, the chylomicrons [2]. However, information concerning possible uptake of exogenous fat particles by the reticuloendothelial system (in particular the Kupffer cells of the liver) is still contradictory [3,4]. In clinical practice, manufactured fat emulsion formulations can be subjected to deliberate modification prior to their administration. For example, in Europe it is becoming established practice to administer parenteral nutrition regimens as a complete mixture comprising fat, amino acids, carbohydrates and electrolytes [5]. Such admixture could lead to changes in the surface properties of the emulsion particles and fo the physical state of the system [6]. At moderate levels of electrolyte the stabilising negative surface charge (about - 40 mV) can be neutralised so that the individual fat particles can be found in a flocculated state. Furthermore, in the presence of high levels of divalent cation (Ca++ or
Fat emulsions have been administered on an acute and chronic basis to follow reticuloendothelial blockade. Flocculated and charge reversed systems have been used to follow the kinetics of deposition and clearance from two organ sites, namely the lung and liver. The rabbit has been used previously as a model to study the deposition of fat emulsions. Thompson and colleagues [9] have shown that it is possible to have high liver uptake of emulsion droplets through sequestration by the reticula-endothelial system. 13
14
STUDIES
MATERIALS
ON THE FATE OF FAT EMULSIONS
FOLLOWING
AND METHOD
Fractionated soybean oil and egg lecithin of a quality suitable for the preparation of intravenous fat emulsions (containing about 75O/,,phosphatidylcholine and about 10”; phosphatidylethanolamine) was obtained as a gift from ICI Pharmaceuticals, UK. Y-sodium (tlj2 = 13.3h) and l~lI-sodium iodide iodide (t/12 =8.ld) were obtained from Harwell Laboratories and Amersham International respectively. Human serum albumin microspheres were obtained as a kit (TCK-9) from CIS (Italy). The wet size of 90% of these particles was stated to be less than 1 pm diameter. The microspheres were labelled with *Tc using 99Tcpertechnetate obtained from a generator (Amersham International). *Tc-labelled antimony sulphide colloid was prepared according to the method of Garzon and colleagues [lo]. Intralipida (10 and 2096) was obtained from KabiVitrum, and consisted of soybean oil emulsified by egg lecithin (1.2OA) with glycerol for isotonicity. The fat particles had a mean diameter (number average) of 250 nm and 305 nm for 1OYb and 200” Intralipid respectively, as measured by Photon Correlation Spectroscopy (Malvern Instruments). Polystyrene latex particles of a reasonably similar mean diameter to Intralipid 20°; (450nm mean diameter) were a gift from Dr J. Hearn, Trent Polytechnic. All other chemicals were of reagent grade quality.
Label&g
of soybean oil
Soybean oil contains unsaturated fatty acids which can be iodinated covalently. Yodine and *3LIodine labelled soybean oil were prepared using the method of Lubran and Pearson [I 11. Soybean oil (1 g) in 6ml ether was mixed with an equal volume of iz31 or ipi1 sodium iodide-thiosulphate solution (3-4 mCi). The mixture was allowed to separate and the lower layer removed. The ethereal solution was washed with water and the ether evaporated at 60°C. About 30% of the iodine was bound to the soybean oil. The binding of the iodine to the oil was checked by administering an emulsion of radiolabelled oil subcutaneously to a group of three rabbits. A control group received labelled sodium iodide solution. The animals were placed on a gamma camera (see below) and imaged over a period of 24 h. Free iodide was cleared rapidly from the injection site (t1/2 = 4.8 * 0.8min) and was localised in the thyroid and bladder, whereas the emulsion was cleared slowly (tlj2 = 13.8 + 1.9h) and no significant accumulation of iodine in the thyroid or bladder was evident in the first 2 h after administration.
INTRAVENOUS
Preparation
ADMINISTRATION
TO RABBITS
of emulsions
Labelled emulsions were prepared using an ultrasonic homogeniser (Dawe Soniprobe). The oil content was 10% and the emulsifier (egg lecithin) was at 1.29;. Glycerol was added to give isotonicity. The particle size of the emulsions was compared with that of Intralipid by using a Coulter Counter TAII with a 50pm orifice tube or by photon correlation spectroscopy (Coulter Nanosizer). In all systems the majority (90”,6) of the particles were below 1 pm in diameter.
Imaging procedure
Labelled colloids were administered to female New Zealand White rabbits (34 kg) via the marginal ear vein. Three rabbits were used in each test and control group. The doses of administered radioactivity were in the range 2-4MBq. The animals were placed on the face of a gamma camera (Searle LFOV or General Electric Maxicamera) fitted with a collimator of appropriate characteristics for the energy of the radionuclide imaged. Dynamic images (45 frames of 20s duration) and static images (60s duration) were taken. The data were recorded on magnetic tape or disc for later computer analysis (Link Gammascope). Regions of interest (liver/spleen, heart/lung etc) were created on computer generated displays and the activity within such regions calculated and corrected for background radiation and radioactive decay. Because of overlying organs it was not possible to create separate regions for the spleen and the heart. In certain studies the circulating level of activity in the blood (i3iI) was measured using a gamma probe
[W Reticuloendothelial
blockade experiments
Polystyrene microspheres. Three rabbits were given 10 ml of a 1096 w/v dispersion of polystyrene latex particles 450nm in diameter, intravenously, 1 h before injection of a 1 ml test dose of albumin microspheres. This dose of microspheres has been shown previously to affect significantly the liver uptake of a test dose of labelled colloid [13] and therefore represented a positive control. Zntralipid. K The effect of single doses of fat emulsion on the reticuloendothelial function was followed by injecting rabbits with 10 ml or 15 ml of 20% Intralipid” 1 h before a test dose of 99 m-technetium labelled albumin microspheres or antimony sulphide colloid. The effect of a longer term infusion of Intralipid” was followed by infusing rabbits with 10% Irmalipid” over a period of 3 days at 2ml emulsion/minute (Saga Instruments Ltd) to give a dose of 6gm/kg/d. On the
CLINICAL
NUTRITION
15
60-
.
1 I
2I
3I
4I
5I
6I
7I Time (min)
8I
9I
10 I
11 I
’ ‘+-E
12
(hr)
The uptake of 99mTc-labelledserum albumin microspheres in the liver: the effect of pre-administration of IntralipidR and polystyrene microspheres. Legend -1 control 0 preadministrationof Intralipid A preadministrationof polystyrene microspheres (300 nm) Fig. 1
last day, 1 h after cessation of the infusion, the animals were given a 1 ml dose of I-123 labelled fat emulsion. Flocculated and charge reversed emulsions. Fat emulsions containing loo, soybean oil labelled with Is11were administered to a group of rabbits in 1 ml doses as the unmodified emulsion (control), a flocculated system containing 5mmol 1 ’ calcium chloride and a charge reversed system containing 20mmol 1-l calcium chloride.
RESULTS Studies on reticuloendothelial
blockade
Single dose of IntraZipid.H The control experiments conducted using 99 m-technetium labelled human serum albumin microspheres as the test colloid showed that in non-blocked animals about 7576 of the administered colloid was taken up rapidly into the liver/spleen region (t1/2 = 60 s) (Fig. 1) with the remaining activity distributed throughout the body. The level in the lung/
heart reached a peak of about 119, shortly after administration and then fell to a plateau level of about 4’+/,(Fig. 2). Following the pre-administration of polystyrene latex particles (positive control) the liver/spleen activity after the dose of test colloid was reduced to 509, (Fig. 1) while the lung/heart activity rose to about 10YOand the peak was absent (Fig. 2). The results following administration of Intralipid” as a potential blocking agent (10 ml of 20°, emulsion) were not significantly different from the untreated control group for both liver/spleen and lung/heart uptake. Similar studies conducted using an alternative test colloid in the form of antimony sulphide colloid (data not shown) also failed to demonstrate any significant difference in body distribution for the Intralipid” treated animals and the untreated control. Prolonged infusion of Intralipid.~’ Preliminary experiments where Intralipida was infused into rabbits at 6g/ kg/day for 3 days did not give rise to a significant difference in the initial distribution pattern for a test dose of 1z31-labelled fat emulsion as compared to animals in the
16
STUDIES ON THE FATE OF FAT EMULSIONS
FOLLOWING
POLYSTYRENE
INTRAVENOUS
ADMINISTRATION
TO RABBITS
MS (300nm)
10I? _= a._ ; 6Y-
INTRALlPtO ‘f
0
ao
4-
‘I&---~ CONTROL
20
I 1
I 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
I 10
Time(min)
I 11
I 12 (hr)
The uptake of PPmTc-labelled serum albumin microspheres in the lung: the effecr of pre-administration of IntralipidK and polystyrene microspheres. Legend as Figure 1 Fig. 2
untreated control group that did not receive a prior infusion.
Studies onjlocculated and charge reversed emulsions The uptake of the labelled fat emulsion into the liver/ spleen and lung/heart regions is shown in Figure 3 for the three different emulsion systems; a control consisting of emulsified soybean oil without added electrolyte and two test systems consisting of emulsions containing added calcium chloride sufficient to give flocculated and charge reversed systems. The uptake of the control emulsion into the liver/spleen followed the expected first order pattern with a half life of about 0.75min (Table 1). The peak level at 5.5min was 47.59,. The uptake in the liver/spleen for the flocculated and charge reversed systems showed different patterns that did not conform to a simple exponential model. The extent of uptake for the two test systems was significantly greater than for the control; 58.5 and 60.5V0 for the flocculated and charge reversed systems respectively. Similarly, the times to peak concentration were extended as compared to the control. At longer times after administration the activity in the liver falls for all systems and the differences between the systems became less marked. After about 50 h similar levels of activity were recorded in the liver/spleen for all three systems. The reduction can be
attributed to metabolism of the oil and release of the label. The three emulsion systems also gave different uptake patterns in the lung/heart region. The control system gave an early peak at about 45 s, while the peak times for flocculated and charge reversed systems were longer. The absolute level of the peak height was higher for the flocculated system than for the control and charge reversed system. The blood concentration-time profiles demonstrated no significant differences in circulating levels of the label. At 60min the blood activity was about 30-400/b of the original level. There was no evidence of observable pathological effects such as respiratory distress.
DISCUSSION Effect of intralipid on REfuncrion The administration of small quantities of Intralipid” (10 or 15 ml) on an acute basis had no significant effect on the subsequent uptake of test colloid particles (albumin microspheres and antimony sulphide colloid) by the live/spleen. This indicates that (i) IntralipidE has no significant blocking effect on RE function at the doses used or (ii) a transient blocking effect occurs which is no longer significant after 1 h or (iii) that the two test colloids are a suitable means for assessing suppressed RE
CLINICAL
NUTRITION
17
Liver
60-
5
10
I
20 50 loo Time (min)
200
500
1000 2000
so00
Lung -_
1
2
5
10
20
50
100
200
500
loo0 2000
5000
Time (min)
Fig. 3 The uptake of “‘I-labelled Legend 0 control A flocculated system ^, _ charge reversed system
emulsions
of soybean oil in the liver and lung.
function produced by polystyrene latex particles but are not suitable for assessing a suppression of the RE system produced by Intralipid.k A compensatory effect of particles diverted from liver to spleen or vice versa would not be revealed by the scintigraphic method. However, this is considered unlikely since the contribution of the spleen to the capture of foreign particles is very small compared to that for the liver. Consequently an attempt was made to block RE
function by repeated dosage of Intralipid* over a period of 3 days, followed by test dose of labelled fat emulsion. Once again (data not shown) no significant differences in organ (liver/spleen, lung/heart) distribution were seen. Therefore it appears that Intralipid* has minimal effects on RE function in the rabbit after single or repeated administration and it is not taken up by the Kupffer cells to any significant extent. These results are
18
STUDIES
ON THE FATE OF FAT EMULSIONS
FOLLOWING
Table 1 Liver and lung uptake of fat emulsions
System Liver Control Flocculated Charge reversed Lung Control Flocculated Charge reversed
Peak Level (“,)
Peak Times (min)
t504, (min)
47.5 58.5 60.5
5.5 10.0 14.2
0.75 1.0 1.6
10.8 12.6 10.8
0.67 1.2 2.8
0.25 0.50 0.45
in agreement with previous studies that have concluded that Intralipide is handled by the body in manner similar to the natural fat particles (the chylomicrons) [14,15].
The fate
of flocculated and charge reversed emulsions
The administration of a labelled flocculated or charge reversed emulsion system to rabbits resulted in significant but transient alterations in the distribution patterns in the liver/spleen and lung/heart regions. More of the emulsion was deposited in the liver/spleen soon after administration if it was given as a flocculated or charge reversed system. Similarly, lung/heart uptake was increased by both systems, and interestingly, for the charge reversed emulsion, the pattern of lung/heart was altered. A flocculated system would be expected to have a higher uptake in the capillary networks of the lung simply by a process of mechanical filtration if the floccules exceeded about 7 pm in size [ 161. With regard to charge reversed systems, an altered pattern of uptake of the particles in the lung/heart region is observed. The literature contains little information on the fate of positively charged colloidal particles [ 17-191 administered intravenously. Certainly it is to be expected that the original positive charge should be altered very rapidly by the adsorption of blood components (probably albumin in the first instance) [20]. Wilkins and Myers [ 171 have reported that polystyrene particles given a positive charge were handled in a different way to similar particles with a negative charge and that the former were taken up first by the lung, before being redistributed to the liver. The same pattern of behaviour appears to occur with the charge reversed emulsions in the present study. It is suggested that a (unknown) positively
blood component
charged
particles
ticles to accumulate
transiently
this is due to an aggregative culated particles
in the lungs.
mechanism
flood capillary
they enter the
agent causes the parWhether
so that the floc-
beds, or a more specific
ADMINISTRATION
TO RABBITS
process of particle adherence is not clear [21]. The process of increased lung uptake lasts only for a short time and after 3-4min the lung/heart levels are the same as the control levels.
CONCLUSIONS The administration of Intralipid’b on an acute and chronic basis to rabbits has no significant effect on reticula-endothelial function. Soybean emulsions labelled with Is11 and emulsified using egg lecithin can demonstrate different liver/spleen and lung/heart uptakes when mixed with electrolyte (calcium chloride) to yield flocculated or charge reversed systems. Consequently flocculated and charge reversed emulsion systems may be used with care in total parenteral nutrition.
REFERENCES PI Judd R H, Freund H, Deckelbaum R J 1984 Human
PI
[31
[41
[51
[61
[71
PI
is taken up onto the
immediately
blood and that this initial coating
INTRAVENOUS
PI
plasma lipoproteins and total parenteral nutrition with intravenous fat emulsions. Journal of Parenteral and Enteral Nutrition 8: 552-555 Erkelens D W, Brunzell J D, Bierman E L 1979 Availability of apolipoprotein CII in relation to the maximal removal capacity for an infused triglyceride emulsion in man, Metabolism 28: 495-501 Van Berkel T J C, Groot P H E, Van To1 A 1984 Interation of the reticuloendothelial system with blood lipid and lipoprotein metabolism, in The ieticuloendothelial system: A Comprehensive Treatise. Vol7A. Phvsiologv. Reichard S M, Filkins T P (eds). Plenum, N;w York305-330 . - . ” Wisse E 1977 Ultrastructure and function of Kupffer cells and other sinusoidal cells in the liver, in Kupffer Cells and Other Sinusoidal Cells. Wisse E, Knook D L (eds), Elsevier/North Holland Biomedical Press, Amsterdam 33-60 Bumham W R, Hansrani P K, Knott C E, Cook J A, Davis S S 1982 Stability of a fat emulsion based intravenous feeding mixture. International Journal of Pharmaceutics 13: 9-22 Davis S S (1983 Stability of intravenous fat emulsions, in Advances in Clinical Nutrition. Johnson I D A (ed) MTP Press, Lancaster 213-239 Dawes W H, Groves M J 1978 The effect of electrolytes on phospholipid-stabilised soybean oil emulsions. International Journal of Pharmaceutics 1: 141-150 Davis S S, Galloway M, Burnham W R, Stevens L 1986 In vitro and clinical studies on intravenous feeding mixtures. Clinical Nutrition 5 21-27 Thompson S W, Jones L D, Ferrell J F, Hunt R D, Meng H C, Kuyama T, Sasaki H, Schaffner F, Singleton W S, Cohn I 1965 Testing of fat emulsions for toxicity. III. Toxicity studies with new fat emulsions and emulsion components. American Journal of Clinical Nutrition 16 43-61
CLINICAL
[lo]
[ 1 l] [12]
[13]
[ 141 [15]
[16]
Garzon 0 L, Palcos M C, Radicella R 1965 A technetium-99m-labelled colloid. International Journal of Applied Radiation and Isotopes 16: 613619 Lubran M, Pearson J D 1958 A screening test for steatorrhoea using 131-I-labelled triolein. Journal of Clinical Pathology 11: 165-169 Illum L, Hardy J G, Davis S S 1982 Gamma ray detection probe for the evaluation of blood activity time profiles. Journal of Pharmacy and Pharmacology 34 Suppl: 91P Illum L, Thomas N W, Davis S S 1986, The effect of a selected suppression of the reticuloendothelial system on the distribution of model carrier particles. Journal of Pharmaceutical Sciences 75: 16-22 Scholler K L 1973 Zur Speicherung verschiedener Fettemulsionen in den Reticuloendothelzellen der Leber, Medizinische Welt 29/30: 1179-l 181 Jarstrand C, Bergham L, Lahnborg G 1978 Human granulocyte and reticuloendothelial system function during Intralipid infusion, Journal of Parenteral and Enteral Nutrition 2: 664-670 Illum L, Davis S S, Wilson C G, Frier M, Hardy J G, Thomas N W 1982 Blood clearance and organ deposition
Submission dare: 21 November
[17]
[18]
[19]
[20]
[21]
NUTRITION
of intravenously administered colloidal particles: the effects of particle size, nature and shape. International Journal of Pharmaceuticals 12: 135-146 Wilkins D J, Myers P A 1966 Studies on the relationship between the electrophoretic properties of colloids and their blood clearance and deposition in the rat. British Journal of Experimental Pathology 47: 568-576 Seno S, Tanaka A, Urata M, Hirata K, Nakatsuka H, Yamamoto S L 1979 Phagocytic response of rat liver capillary endothelial cells and Kupffer cells to positive and negatively charged ion colloid particles. Cell Structure Funct. 1: 119-128 Caride V J, Zaret B L 1977 Liposome accumulation in regions of experimental myocardial infarction. Science 198: 735-738 Davis S S, Douglas S J, Illum L, Jones l’ D E, Mak E, Muller R 1986 Passive and active delivery of colloidal carriers and the role of surface modifications, in Targeting of Drugs with Synthetic Systems. Gregoriadis G, Senior J (eds) Plenum Press, London, 123-146 Abra R M, Hunt CA, Lau D T 1984, Liposome deposition in viva. VI. Delivery to the lung. Journal of Pharmaceutical Sciences 73: 203-206
1985. Accepted after revision: 21 June 1986
19
NUTRITION
(1987) 6: 13-19
Studies on the Fate ofFat Emulsions Following Intravenous Administration to Rabbits and thk Effect of Added Electrolyte S. S. Davis*, L. Illumt,
P. West* and M. Galloway*
*Department of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK. tDepartment of Pharmaceutics, Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, (Reprint requests to S.S.D.)
Denmark.
The effect of the fat emulsion Intralipid$+ (soybean oil emulsified with egg lecithin) on ABSTRACT reticuloendothelial function has been evaluated in rabbits using the non-invasive technique of gamma scintigraphy. Neither acute nor chronic administration of the fat emulsion had any significant effect on the subsequent uptake of labelled test colloids (microspheres and fat emulsions) into the liver and spleen. Studies were also made on the uptake of labelled fat emulsions that had modified physical properties as caused by the addition of electrolyte (calcium chloride). Flocculated emulsions and those carrying a positive charge (charge reversed) demonstrated different patterns of uptake in the liver/spleen and heart/lung regions of the rabbit immediately after intravenous administration. The results are discussed in terms of entrappment of emulsion Aoccules in the capillary beds of the lungs and biological
fate of positively
charged
colloidal
particles.
Mg+ + greater than about 10 mmol 1 -I) the fat particles can even carry a net positive charge (+ 15 mV) [‘7]. The effects of these physical changes on the in vivo behaviour of fat emulsions had nof been studied in detail. Apparently it is assumed that any effect will be reversed easily upon dilution of the mixed emulsion when injected into the blood. This assumption may not be valid since the adsorption of blood components onto emulsion droplets occurs very rapidly [8] and therefore the possibility exists of an enhanced state of flocculation of droplets in vivo or an altered adsorption of blood components (e.g. type of apoprotein). Therefore, we have conducted a series of studies using the non-invasive technique of gamma scintigraphy to follow the whole body deposition of fat emulsions and thereby to evaluate the effect on reticuloendothelial function. The rabbit has been used as the animal model.
INTRODUCTION Emulsions prepared from vegetable oils such as soybean oil (and more recently sesame oil) emulsified with egg or soy phosphatides, have been used with success in parenteral nutrition with remarkably few adverse reactions and side effects 111. Their fate in the body, as determined by the adsorption of apolipoproteins and subsequent clearance from the blood, has been found similar to that of the endogenous lipid particles, the chylomicrons [2]. However, information concerning possible uptake of exogenous fat particles by the reticuloendothelial system (in particular the Kupffer cells of the liver) is still contradictory [3,4]. In clinical practice, manufactured fat emulsion formulations can be subjected to deliberate modification prior to their administration. For example, in Europe it is becoming established practice to administer parenteral nutrition regimens as a complete mixture comprising fat, amino acids, carbohydrates and electrolytes [5]. Such admixture could lead to changes in the surface properties of the emulsion particles and fo the physical state of the system [6]. At moderate levels of electrolyte the stabilising negative surface charge (about - 40 mV) can be neutralised so that the individual fat particles can be found in a flocculated state. Furthermore, in the presence of high levels of divalent cation (Ca++ or
Fat emulsions have been administered on an acute and chronic basis to follow reticuloendothelial blockade. Flocculated and charge reversed systems have been used to follow the kinetics of deposition and clearance from two organ sites, namely the lung and liver. The rabbit has been used previously as a model to study the deposition of fat emulsions. Thompson and colleagues [9] have shown that it is possible to have high liver uptake of emulsion droplets through sequestration by the reticula-endothelial system. 13
14
STUDIES
MATERIALS
ON THE FATE OF FAT EMULSIONS
FOLLOWING
AND METHOD
Fractionated soybean oil and egg lecithin of a quality suitable for the preparation of intravenous fat emulsions (containing about 75O/,,phosphatidylcholine and about 10”; phosphatidylethanolamine) was obtained as a gift from ICI Pharmaceuticals, UK. Y-sodium (tlj2 = 13.3h) and l~lI-sodium iodide iodide (t/12 =8.ld) were obtained from Harwell Laboratories and Amersham International respectively. Human serum albumin microspheres were obtained as a kit (TCK-9) from CIS (Italy). The wet size of 90% of these particles was stated to be less than 1 pm diameter. The microspheres were labelled with *Tc using 99Tcpertechnetate obtained from a generator (Amersham International). *Tc-labelled antimony sulphide colloid was prepared according to the method of Garzon and colleagues [lo]. Intralipida (10 and 2096) was obtained from KabiVitrum, and consisted of soybean oil emulsified by egg lecithin (1.2OA) with glycerol for isotonicity. The fat particles had a mean diameter (number average) of 250 nm and 305 nm for 1OYb and 200” Intralipid respectively, as measured by Photon Correlation Spectroscopy (Malvern Instruments). Polystyrene latex particles of a reasonably similar mean diameter to Intralipid 20°; (450nm mean diameter) were a gift from Dr J. Hearn, Trent Polytechnic. All other chemicals were of reagent grade quality.
Label&g
of soybean oil
Soybean oil contains unsaturated fatty acids which can be iodinated covalently. Yodine and *3LIodine labelled soybean oil were prepared using the method of Lubran and Pearson [I 11. Soybean oil (1 g) in 6ml ether was mixed with an equal volume of iz31 or ipi1 sodium iodide-thiosulphate solution (3-4 mCi). The mixture was allowed to separate and the lower layer removed. The ethereal solution was washed with water and the ether evaporated at 60°C. About 30% of the iodine was bound to the soybean oil. The binding of the iodine to the oil was checked by administering an emulsion of radiolabelled oil subcutaneously to a group of three rabbits. A control group received labelled sodium iodide solution. The animals were placed on a gamma camera (see below) and imaged over a period of 24 h. Free iodide was cleared rapidly from the injection site (t1/2 = 4.8 * 0.8min) and was localised in the thyroid and bladder, whereas the emulsion was cleared slowly (tlj2 = 13.8 + 1.9h) and no significant accumulation of iodine in the thyroid or bladder was evident in the first 2 h after administration.
INTRAVENOUS
Preparation
ADMINISTRATION
TO RABBITS
of emulsions
Labelled emulsions were prepared using an ultrasonic homogeniser (Dawe Soniprobe). The oil content was 10% and the emulsifier (egg lecithin) was at 1.29;. Glycerol was added to give isotonicity. The particle size of the emulsions was compared with that of Intralipid by using a Coulter Counter TAII with a 50pm orifice tube or by photon correlation spectroscopy (Coulter Nanosizer). In all systems the majority (90”,6) of the particles were below 1 pm in diameter.
Imaging procedure
Labelled colloids were administered to female New Zealand White rabbits (34 kg) via the marginal ear vein. Three rabbits were used in each test and control group. The doses of administered radioactivity were in the range 2-4MBq. The animals were placed on the face of a gamma camera (Searle LFOV or General Electric Maxicamera) fitted with a collimator of appropriate characteristics for the energy of the radionuclide imaged. Dynamic images (45 frames of 20s duration) and static images (60s duration) were taken. The data were recorded on magnetic tape or disc for later computer analysis (Link Gammascope). Regions of interest (liver/spleen, heart/lung etc) were created on computer generated displays and the activity within such regions calculated and corrected for background radiation and radioactive decay. Because of overlying organs it was not possible to create separate regions for the spleen and the heart. In certain studies the circulating level of activity in the blood (i3iI) was measured using a gamma probe
[W Reticuloendothelial
blockade experiments
Polystyrene microspheres. Three rabbits were given 10 ml of a 1096 w/v dispersion of polystyrene latex particles 450nm in diameter, intravenously, 1 h before injection of a 1 ml test dose of albumin microspheres. This dose of microspheres has been shown previously to affect significantly the liver uptake of a test dose of labelled colloid [13] and therefore represented a positive control. Zntralipid. K The effect of single doses of fat emulsion on the reticuloendothelial function was followed by injecting rabbits with 10 ml or 15 ml of 20% Intralipid” 1 h before a test dose of 99 m-technetium labelled albumin microspheres or antimony sulphide colloid. The effect of a longer term infusion of Intralipid” was followed by infusing rabbits with 10% Irmalipid” over a period of 3 days at 2ml emulsion/minute (Saga Instruments Ltd) to give a dose of 6gm/kg/d. On the
CLINICAL
NUTRITION
15
60-
.
1 I
2I
3I
4I
5I
6I
7I Time (min)
8I
9I
10 I
11 I
’ ‘+-E
12
(hr)
The uptake of 99mTc-labelledserum albumin microspheres in the liver: the effect of pre-administration of IntralipidR and polystyrene microspheres. Legend -1 control 0 preadministrationof Intralipid A preadministrationof polystyrene microspheres (300 nm) Fig. 1
last day, 1 h after cessation of the infusion, the animals were given a 1 ml dose of I-123 labelled fat emulsion. Flocculated and charge reversed emulsions. Fat emulsions containing loo, soybean oil labelled with Is11were administered to a group of rabbits in 1 ml doses as the unmodified emulsion (control), a flocculated system containing 5mmol 1 ’ calcium chloride and a charge reversed system containing 20mmol 1-l calcium chloride.
RESULTS Studies on reticuloendothelial
blockade
Single dose of IntraZipid.H The control experiments conducted using 99 m-technetium labelled human serum albumin microspheres as the test colloid showed that in non-blocked animals about 7576 of the administered colloid was taken up rapidly into the liver/spleen region (t1/2 = 60 s) (Fig. 1) with the remaining activity distributed throughout the body. The level in the lung/
heart reached a peak of about 119, shortly after administration and then fell to a plateau level of about 4’+/,(Fig. 2). Following the pre-administration of polystyrene latex particles (positive control) the liver/spleen activity after the dose of test colloid was reduced to 509, (Fig. 1) while the lung/heart activity rose to about 10YOand the peak was absent (Fig. 2). The results following administration of Intralipid” as a potential blocking agent (10 ml of 20°, emulsion) were not significantly different from the untreated control group for both liver/spleen and lung/heart uptake. Similar studies conducted using an alternative test colloid in the form of antimony sulphide colloid (data not shown) also failed to demonstrate any significant difference in body distribution for the Intralipid” treated animals and the untreated control. Prolonged infusion of Intralipid.~’ Preliminary experiments where Intralipida was infused into rabbits at 6g/ kg/day for 3 days did not give rise to a significant difference in the initial distribution pattern for a test dose of 1z31-labelled fat emulsion as compared to animals in the
16
STUDIES ON THE FATE OF FAT EMULSIONS
FOLLOWING
POLYSTYRENE
INTRAVENOUS
ADMINISTRATION
TO RABBITS
MS (300nm)
10I? _= a._ ; 6Y-
INTRALlPtO ‘f
0
ao
4-
‘I&---~ CONTROL
20
I 1
I 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
I 10
Time(min)
I 11
I 12 (hr)
The uptake of PPmTc-labelled serum albumin microspheres in the lung: the effecr of pre-administration of IntralipidK and polystyrene microspheres. Legend as Figure 1 Fig. 2
untreated control group that did not receive a prior infusion.
Studies onjlocculated and charge reversed emulsions The uptake of the labelled fat emulsion into the liver/ spleen and lung/heart regions is shown in Figure 3 for the three different emulsion systems; a control consisting of emulsified soybean oil without added electrolyte and two test systems consisting of emulsions containing added calcium chloride sufficient to give flocculated and charge reversed systems. The uptake of the control emulsion into the liver/spleen followed the expected first order pattern with a half life of about 0.75min (Table 1). The peak level at 5.5min was 47.59,. The uptake in the liver/spleen for the flocculated and charge reversed systems showed different patterns that did not conform to a simple exponential model. The extent of uptake for the two test systems was significantly greater than for the control; 58.5 and 60.5V0 for the flocculated and charge reversed systems respectively. Similarly, the times to peak concentration were extended as compared to the control. At longer times after administration the activity in the liver falls for all systems and the differences between the systems became less marked. After about 50 h similar levels of activity were recorded in the liver/spleen for all three systems. The reduction can be
attributed to metabolism of the oil and release of the label. The three emulsion systems also gave different uptake patterns in the lung/heart region. The control system gave an early peak at about 45 s, while the peak times for flocculated and charge reversed systems were longer. The absolute level of the peak height was higher for the flocculated system than for the control and charge reversed system. The blood concentration-time profiles demonstrated no significant differences in circulating levels of the label. At 60min the blood activity was about 30-400/b of the original level. There was no evidence of observable pathological effects such as respiratory distress.
DISCUSSION Effect of intralipid on REfuncrion The administration of small quantities of Intralipid” (10 or 15 ml) on an acute basis had no significant effect on the subsequent uptake of test colloid particles (albumin microspheres and antimony sulphide colloid) by the live/spleen. This indicates that (i) IntralipidE has no significant blocking effect on RE function at the doses used or (ii) a transient blocking effect occurs which is no longer significant after 1 h or (iii) that the two test colloids are a suitable means for assessing suppressed RE
CLINICAL
NUTRITION
17
Liver
60-
5
10
I
20 50 loo Time (min)
200
500
1000 2000
so00
Lung -_
1
2
5
10
20
50
100
200
500
loo0 2000
5000
Time (min)
Fig. 3 The uptake of “‘I-labelled Legend 0 control A flocculated system ^, _ charge reversed system
emulsions
of soybean oil in the liver and lung.
function produced by polystyrene latex particles but are not suitable for assessing a suppression of the RE system produced by Intralipid.k A compensatory effect of particles diverted from liver to spleen or vice versa would not be revealed by the scintigraphic method. However, this is considered unlikely since the contribution of the spleen to the capture of foreign particles is very small compared to that for the liver. Consequently an attempt was made to block RE
function by repeated dosage of Intralipid* over a period of 3 days, followed by test dose of labelled fat emulsion. Once again (data not shown) no significant differences in organ (liver/spleen, lung/heart) distribution were seen. Therefore it appears that Intralipid* has minimal effects on RE function in the rabbit after single or repeated administration and it is not taken up by the Kupffer cells to any significant extent. These results are
18
STUDIES
ON THE FATE OF FAT EMULSIONS
FOLLOWING
Table 1 Liver and lung uptake of fat emulsions
System Liver Control Flocculated Charge reversed Lung Control Flocculated Charge reversed
Peak Level (“,)
Peak Times (min)
t504, (min)
47.5 58.5 60.5
5.5 10.0 14.2
0.75 1.0 1.6
10.8 12.6 10.8
0.67 1.2 2.8
0.25 0.50 0.45
in agreement with previous studies that have concluded that Intralipide is handled by the body in manner similar to the natural fat particles (the chylomicrons) [14,15].
The fate
of flocculated and charge reversed emulsions
The administration of a labelled flocculated or charge reversed emulsion system to rabbits resulted in significant but transient alterations in the distribution patterns in the liver/spleen and lung/heart regions. More of the emulsion was deposited in the liver/spleen soon after administration if it was given as a flocculated or charge reversed system. Similarly, lung/heart uptake was increased by both systems, and interestingly, for the charge reversed emulsion, the pattern of lung/heart was altered. A flocculated system would be expected to have a higher uptake in the capillary networks of the lung simply by a process of mechanical filtration if the floccules exceeded about 7 pm in size [ 161. With regard to charge reversed systems, an altered pattern of uptake of the particles in the lung/heart region is observed. The literature contains little information on the fate of positively charged colloidal particles [ 17-191 administered intravenously. Certainly it is to be expected that the original positive charge should be altered very rapidly by the adsorption of blood components (probably albumin in the first instance) [20]. Wilkins and Myers [ 171 have reported that polystyrene particles given a positive charge were handled in a different way to similar particles with a negative charge and that the former were taken up first by the lung, before being redistributed to the liver. The same pattern of behaviour appears to occur with the charge reversed emulsions in the present study. It is suggested that a (unknown) positively
blood component
charged
particles
ticles to accumulate
transiently
this is due to an aggregative culated particles
in the lungs.
mechanism
flood capillary
they enter the
agent causes the parWhether
so that the floc-
beds, or a more specific
ADMINISTRATION
TO RABBITS
process of particle adherence is not clear [21]. The process of increased lung uptake lasts only for a short time and after 3-4min the lung/heart levels are the same as the control levels.
CONCLUSIONS The administration of Intralipid’b on an acute and chronic basis to rabbits has no significant effect on reticula-endothelial function. Soybean emulsions labelled with Is11 and emulsified using egg lecithin can demonstrate different liver/spleen and lung/heart uptakes when mixed with electrolyte (calcium chloride) to yield flocculated or charge reversed systems. Consequently flocculated and charge reversed emulsion systems may be used with care in total parenteral nutrition.
REFERENCES PI Judd R H, Freund H, Deckelbaum R J 1984 Human
PI
[31
[41
[51
[61
[71
PI
is taken up onto the
immediately
blood and that this initial coating
INTRAVENOUS
PI
plasma lipoproteins and total parenteral nutrition with intravenous fat emulsions. Journal of Parenteral and Enteral Nutrition 8: 552-555 Erkelens D W, Brunzell J D, Bierman E L 1979 Availability of apolipoprotein CII in relation to the maximal removal capacity for an infused triglyceride emulsion in man, Metabolism 28: 495-501 Van Berkel T J C, Groot P H E, Van To1 A 1984 Interation of the reticuloendothelial system with blood lipid and lipoprotein metabolism, in The ieticuloendothelial system: A Comprehensive Treatise. Vol7A. Phvsiologv. Reichard S M, Filkins T P (eds). Plenum, N;w York305-330 . - . ” Wisse E 1977 Ultrastructure and function of Kupffer cells and other sinusoidal cells in the liver, in Kupffer Cells and Other Sinusoidal Cells. Wisse E, Knook D L (eds), Elsevier/North Holland Biomedical Press, Amsterdam 33-60 Bumham W R, Hansrani P K, Knott C E, Cook J A, Davis S S 1982 Stability of a fat emulsion based intravenous feeding mixture. International Journal of Pharmaceutics 13: 9-22 Davis S S (1983 Stability of intravenous fat emulsions, in Advances in Clinical Nutrition. Johnson I D A (ed) MTP Press, Lancaster 213-239 Dawes W H, Groves M J 1978 The effect of electrolytes on phospholipid-stabilised soybean oil emulsions. International Journal of Pharmaceutics 1: 141-150 Davis S S, Galloway M, Burnham W R, Stevens L 1986 In vitro and clinical studies on intravenous feeding mixtures. Clinical Nutrition 5 21-27 Thompson S W, Jones L D, Ferrell J F, Hunt R D, Meng H C, Kuyama T, Sasaki H, Schaffner F, Singleton W S, Cohn I 1965 Testing of fat emulsions for toxicity. III. Toxicity studies with new fat emulsions and emulsion components. American Journal of Clinical Nutrition 16 43-61
CLINICAL
[lo]
[ 1 l] [12]
[13]
[ 141 [15]
[16]
Garzon 0 L, Palcos M C, Radicella R 1965 A technetium-99m-labelled colloid. International Journal of Applied Radiation and Isotopes 16: 613619 Lubran M, Pearson J D 1958 A screening test for steatorrhoea using 131-I-labelled triolein. Journal of Clinical Pathology 11: 165-169 Illum L, Hardy J G, Davis S S 1982 Gamma ray detection probe for the evaluation of blood activity time profiles. Journal of Pharmacy and Pharmacology 34 Suppl: 91P Illum L, Thomas N W, Davis S S 1986, The effect of a selected suppression of the reticuloendothelial system on the distribution of model carrier particles. Journal of Pharmaceutical Sciences 75: 16-22 Scholler K L 1973 Zur Speicherung verschiedener Fettemulsionen in den Reticuloendothelzellen der Leber, Medizinische Welt 29/30: 1179-l 181 Jarstrand C, Bergham L, Lahnborg G 1978 Human granulocyte and reticuloendothelial system function during Intralipid infusion, Journal of Parenteral and Enteral Nutrition 2: 664-670 Illum L, Davis S S, Wilson C G, Frier M, Hardy J G, Thomas N W 1982 Blood clearance and organ deposition
Submission dare: 21 November
[17]
[18]
[19]
[20]
[21]
NUTRITION
of intravenously administered colloidal particles: the effects of particle size, nature and shape. International Journal of Pharmaceuticals 12: 135-146 Wilkins D J, Myers P A 1966 Studies on the relationship between the electrophoretic properties of colloids and their blood clearance and deposition in the rat. British Journal of Experimental Pathology 47: 568-576 Seno S, Tanaka A, Urata M, Hirata K, Nakatsuka H, Yamamoto S L 1979 Phagocytic response of rat liver capillary endothelial cells and Kupffer cells to positive and negatively charged ion colloid particles. Cell Structure Funct. 1: 119-128 Caride V J, Zaret B L 1977 Liposome accumulation in regions of experimental myocardial infarction. Science 198: 735-738 Davis S S, Douglas S J, Illum L, Jones l’ D E, Mak E, Muller R 1986 Passive and active delivery of colloidal carriers and the role of surface modifications, in Targeting of Drugs with Synthetic Systems. Gregoriadis G, Senior J (eds) Plenum Press, London, 123-146 Abra R M, Hunt CA, Lau D T 1984, Liposome deposition in viva. VI. Delivery to the lung. Journal of Pharmaceutical Sciences 73: 203-206
1985. Accepted after revision: 21 June 1986
19