Intralipid
Adversely By Schmuel
Affects Reticuloendothelial Katz, Brian R. Plaisier,
Walter
J. Folkening,
Bacterial
Clearance
and Jay L. Grosfeld
Indianapolis, Indiana 0 Lipid emulsion is a major caloric source in patients receiving total parenteral nutrition. Cleared by the Kuppfer cells of the reticuloendothelial system (RES), lipid emulsion may adversely affect the RES function by decreasing its ability to remove blood-borne bacteria. This study evaluates and compares the blood clearance and organ localization of viable radiolabeled [“S] Escherichia co/i following slow intraperitoneal (IP) and more rapid intravenous (IV) administration of a 20% fat emulsion (FE). Sixty male SpragueDawley rats weighing 150 g were placed in 6 experimental groups (10 rats per group). Group I received IP normal saline (3 mL/d for 3 days); group II received IP FE (20%) (4 g/kg/d for 3 days); and group Ill received normal saline IV (3 mL/d for 3 days). The remaining animals received a slow (15. minute) IV infusion of FE (4 g/kg/dose) prior to bacterial challenge: group IV at 4 hours; group V at 24 hours; and group IV at 4 and 24 hours. E co/i {lO*/mL) were injected via the tail vein. Blood samples were obtained for clearance study. At 10 minutes, tissue samples (50 to 100 mg) of liver, spleen, kidney, and lung were obtained and processed for liquid scintillation counting. Although rapid bacterial blood clearance was found in all the groups, there was a significant change in organ localization of bacteria. Normal distribution of bacteria in group I was as follows: liver 70.1% 2 6.2%, spleen 5.2% * 1.2%. kidney 0.2% 2 0.04%. and lung 1.6% -r0.6%. There was a slight increase in lung localization of bacteria in rats receiving IP FE (3.7% 2 1.5%; P < .05). There was a significant increase in lung trapping of bacteria in all the groups receiving IV FE and a decreased liver localization of bacteria at 24 (group V) and 4 and 24 hours (group VI) following administration of FE. These data suggest that IV FE decreases hepatic phagocytosis, increases pulmonary localization of Eco/& and may result in increased susceptibility to infection. These observations suggest that FE emulsion should be cautiously administered in regard to dose and rate in septic patients. Copyright 6 1991 by W.B. Saunders Company INDEX WORDS: Parenteral nutrition, fat emulsion; reticuloendothelial system, bacterial clearance.
F
AT EMULSIONS are frequently used in wellbalanced parenteral nutrition as a major source of calories and fatty acids.1-3 In general, patients tolerate the currently used formula consisting of soya oil, phospholipid from egg yolk, and glycerol quite well. However, complications such as lung and liver fatty deposits, fatty microembolism, bilirubinemia, and cholestasis have been reported with lipid administration.“’ It has been suggested that some of these complications might be dose or rate related. The accumulation of “oil-droplets” in the liver can inhibit bacterial clearance by the reticuloendothelial system (RES).7 This RES dysfunction may result in increased susceptibility to infection. This study evaluates and compares the blood clearance of viable radiolabled Journalof Pediatric
Surgery, Vol26, No 8 (August), 1991: pp 921-924
[35S] Escherichiu
coli following slow intraperitoneal (II’) and more rapid intravenous (IV) administration of a 20% FE. MATERIALS
AND
METHODS
Experimental Animals Sixtymale Sprague-Dawley rats (Harlan SD Inc, Indianapolis, IN) weighing 140 to 150 g were housed four per cage and fed with standard Rodent Laboratory Chow (no. 5001; Purina Mills Inc, St Louis, MO) and tap water ad libitum. For IV injection or blood sampling, the rats were sedated using intramuscular (IM) ketamine “cocktail” (0.1 mL/rat; containing 100 mg ketamine, 2.2 mg promazine, and 0.4 mg atropine per mL). The tail was soaked in warm water (50°C) and cleansed. A Butterfly Infusion Set (27gauge, g-in tubing, no. 4395; Abbott Hospital Inc, North Chicago, IL) was used for blood drawing and IV injection. The rats were placed into 6 groups according to the mode and rate of infusion of 20% FE (McGawNutralipid, 20% IV Fat Emulsion; The Green Cross Carp, Osaka, Japan). Group I received IP injection of normal saline (3 mud for 3 days); group II received IP injection of 4 g/kg/d of FE for 3 days; group III received IV injection of normal saline (3 mL/d for 3 days). Groups IV, V. and VI received IV injection of FE (4 g/kg/dose) over 15 minutes, at 4 hours, 24 hours, and 4 and 24 hours, respectively, prior to bacterial challenge. Radiolabeling of Bacteria The procedures used for radiolabeling of bacteria have been recently reported in detail” and will be briefly described: E coli (clinical isolate; Indiana University Hospital, Indianapolis, IN) was cultured to late exponential phase in a chemically defined medium containing 5 pXi/mL [“S] Na,SO, (New England Nuclear, Boston, Ma). “S-labeled bacteria were harvested and washed. Bacterial concentration was adjusted spectrophotometrically to approximately lo9 viable E coli per milliliter. Samples of the suspension were obtained for radioactivity determination prior to use.
Bacterial Clearance and Organ Localization For bacterial blood clearance and organ localization studies, the animals were sedated with an IM injection of ketamine cocktail and kept in a restraining cage. One milliliter of ?S-labeled viable E coli (lo9 bacteria/ml) was injected via the tail vein. For clearance, blood samples (50 kL) were collected using heparinized micropipettes into scintillation glass vials at 1,5, and 10 minutes following
From the Section of Pediatric Surgery, Department of Surgery, and the Department of Microbiology and Immunology, Indiana University School of Medicine, and the James Whitcomb Riley Hospital for Children, Indianapolis, IN. Presented at the Jens G. Rosenkrantz Resident Competition at the 42nd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Boston, Massachusetts, October 6-7, 1990. Address reprint requests to Jay L. Grosfeld, MD, Surgeon-in-Chief, J. W: Riley Hospital for Children, 702 Bamhill Dr (K-21) Indianapolis, IN 46202-5200. Copyright 0 1991 by W.B. Saunders Company 0022-3468/91/2608-0008$03.00!0 921
KATZ ET AL
922
bacterial challenge. At 10 minutes, the liver, spleen, kidney, and lung were immediately removed. Small tissue samples (50 to 100 mg) of the liver, spleen, kidney, and lungs as well as the blood samples were processed for liquid scintillation counting and expressed in disintegrations per minute (dpm). The final distribution of bacteria in the different organs was calculated from the input specific radioactivity (dpm/bacteria) and expressed as percentage of the injected bacteria per gram of tissue and per total organ. The experimental groups were studied concurrently with controls using the same bacterial concentration at 4,24, and 4 and 24 hours following administration of FE.
X OF INJECTED
60
LIVER
Radioactivity Determination Samples of cultured bacteria, blood, and tissue were dried and dissolved by treatment of 1.0 mL of 100% Soluene 350 (Packard Camberra Co, Downers Grove, IL) tissue solubilizer overnight at 50°C. The dissolved tissues were dried by 100% isopropyl alcohol and decolorized by 30% H,O, for 30 minutes and cooled to room temperature. Fifteen milliliters of Insta-Gel scintillation fluid (Packard Instrument Co, Downers Grove, IL) was added. Radioactivity was determined using a 2200 CA Tricarb analyzer (Packard Instrument Co, Downers Grove, IL) interfaced with an IBM microcomputer. Data were corrected for quench using a calibration curve determined by external standardization, and radioactivity was expressed as ‘5Sdpm.
Biochemistry Blood samples for serum cholesterol, free fatty acid (FFA), and triglycerides were obtained from 3 rats in each experimental group at the time of bacterial challenge.
Statistical Analysis Results were expressed as mean percent of injected bacteria f standard deviation. The differences in blood clearance and organ localization of bacteria were evaluated for validity by one-way analysis ofvariance(ANOVA) and the Student’s t test. RESULTS
There was no mortality following administration of IP and IV FE. The serum levels of cholesterol, FFA, and triglycerides are summarized in Table 1. There was no significant difference in these parameters in rats following IP injection compared with controls. Significant increase in blood levels of FFAs and triglycerides were found in rats at 4 (group IV) and 4 and 24 (group VI) hours following IV administration of FE.
Table 1. Serum Triglycerides. Free Fatty Acids, and Cholesterol in Flats Triglycerides Group
Control IP
Free Fatty Acids (kmol/L)
110 f 18
550 -c 70
65?
75 * 20
518 2 65
87 + 17 77 % 14
IV 4 h
800 f 95
2200 lr 350
IV24h
110 t 23
770 2 90
1350 f 260
2460 2 300
IV4and24h
Cholesterol
(mg/dL)
NOTE. n = 3 in each group. Abbreviations: IP, intraperitoneal; IV, intravenous.
MgldL)
72?
10
16
133 + 42
BACTERIA
60
SPLEEN
KIDNEY
LUNG
Fig 1. Comparative liver, spleen, kidney, and lung localization of IV injected radiolabeled E co/i in rats following IP injection of normal saline (n = 10) (D) and FE (a) 20% for 3 days. Values represent mean percent of injected bacteria (dpm/organ). Note a significant increase in lung localization of bacteria in FE rats.
Bacterial Clearance and Oqan Localization
IV injection of viable radiolabeled E coli was followed by immediate clearance of bacteria from the blood; 95% of the injected bacteria disappeared from the blood stream in the first 5 minutes. There was no significant change in the clearance rate of viable bacteria among the experimental groups. However, administration of FE did affect bacterial localization in the different organs. IP injection for 3 days results in an increase in lung localization of bacteria (3.7% + 1.5% v control 1.6% + 0.56%; P < .05) with no change in the ability of the liver to trap bacteria (Fig 1). IV administration of FE was followed by an increase in lung localization of bacteria in all the rats. Reduced bacterial trapping by the liver was found in rats at 24 hours (group V) and 4 and 24 hours (group VI) following IV administration of FE (Fig 2). DISCUSSION
The importance of aggressive nutritional support in improving host defenses and decreasing the risk of morbid infection has been clearly documented. Theoretically, FE being a major source of energy, is expected to further improve the resistance to infection. Paradoxically, it has been shown that “lipid overload” may result in various systemic complications. When the rate of lipid infusion exceeds the capacity of capillary endothelial lipoprotein lipase to metabolize lipid-emulsion triglycerides, the excess lipid may either coat, or be engulfed by, phagocytes and macrophages interfering with their normal function.4x9.10 Nugent” demonstrated that in vitro intralipid reduced phagocytic and pinocytic activities of mouse peritoneal macrophages. In contrast, adding FE to the parenteral nutrition regimen in cancer patients did not affect neutrophil chemotaxis and bacteriotidal activity.‘* Most of the data concerning the effect of FE on host immune response were obtained in in
INTRALIPID AND RES BACTERIAL CLEARANCE
% OF INJECTED
923
BACTERIA
* **
P-Eon5
P
Adversely By Schmuel
Affects Reticuloendothelial Katz, Brian R. Plaisier,
Walter
J. Folkening,
Bacterial
Clearance
and Jay L. Grosfeld
Indianapolis, Indiana 0 Lipid emulsion is a major caloric source in patients receiving total parenteral nutrition. Cleared by the Kuppfer cells of the reticuloendothelial system (RES), lipid emulsion may adversely affect the RES function by decreasing its ability to remove blood-borne bacteria. This study evaluates and compares the blood clearance and organ localization of viable radiolabeled [“S] Escherichia co/i following slow intraperitoneal (IP) and more rapid intravenous (IV) administration of a 20% fat emulsion (FE). Sixty male SpragueDawley rats weighing 150 g were placed in 6 experimental groups (10 rats per group). Group I received IP normal saline (3 mL/d for 3 days); group II received IP FE (20%) (4 g/kg/d for 3 days); and group Ill received normal saline IV (3 mL/d for 3 days). The remaining animals received a slow (15. minute) IV infusion of FE (4 g/kg/dose) prior to bacterial challenge: group IV at 4 hours; group V at 24 hours; and group IV at 4 and 24 hours. E co/i {lO*/mL) were injected via the tail vein. Blood samples were obtained for clearance study. At 10 minutes, tissue samples (50 to 100 mg) of liver, spleen, kidney, and lung were obtained and processed for liquid scintillation counting. Although rapid bacterial blood clearance was found in all the groups, there was a significant change in organ localization of bacteria. Normal distribution of bacteria in group I was as follows: liver 70.1% 2 6.2%, spleen 5.2% * 1.2%. kidney 0.2% 2 0.04%. and lung 1.6% -r0.6%. There was a slight increase in lung localization of bacteria in rats receiving IP FE (3.7% 2 1.5%; P < .05). There was a significant increase in lung trapping of bacteria in all the groups receiving IV FE and a decreased liver localization of bacteria at 24 (group V) and 4 and 24 hours (group VI) following administration of FE. These data suggest that IV FE decreases hepatic phagocytosis, increases pulmonary localization of Eco/& and may result in increased susceptibility to infection. These observations suggest that FE emulsion should be cautiously administered in regard to dose and rate in septic patients. Copyright 6 1991 by W.B. Saunders Company INDEX WORDS: Parenteral nutrition, fat emulsion; reticuloendothelial system, bacterial clearance.
F
AT EMULSIONS are frequently used in wellbalanced parenteral nutrition as a major source of calories and fatty acids.1-3 In general, patients tolerate the currently used formula consisting of soya oil, phospholipid from egg yolk, and glycerol quite well. However, complications such as lung and liver fatty deposits, fatty microembolism, bilirubinemia, and cholestasis have been reported with lipid administration.“’ It has been suggested that some of these complications might be dose or rate related. The accumulation of “oil-droplets” in the liver can inhibit bacterial clearance by the reticuloendothelial system (RES).7 This RES dysfunction may result in increased susceptibility to infection. This study evaluates and compares the blood clearance of viable radiolabled Journalof Pediatric
Surgery, Vol26, No 8 (August), 1991: pp 921-924
[35S] Escherichiu
coli following slow intraperitoneal (II’) and more rapid intravenous (IV) administration of a 20% FE. MATERIALS
AND
METHODS
Experimental Animals Sixtymale Sprague-Dawley rats (Harlan SD Inc, Indianapolis, IN) weighing 140 to 150 g were housed four per cage and fed with standard Rodent Laboratory Chow (no. 5001; Purina Mills Inc, St Louis, MO) and tap water ad libitum. For IV injection or blood sampling, the rats were sedated using intramuscular (IM) ketamine “cocktail” (0.1 mL/rat; containing 100 mg ketamine, 2.2 mg promazine, and 0.4 mg atropine per mL). The tail was soaked in warm water (50°C) and cleansed. A Butterfly Infusion Set (27gauge, g-in tubing, no. 4395; Abbott Hospital Inc, North Chicago, IL) was used for blood drawing and IV injection. The rats were placed into 6 groups according to the mode and rate of infusion of 20% FE (McGawNutralipid, 20% IV Fat Emulsion; The Green Cross Carp, Osaka, Japan). Group I received IP injection of normal saline (3 mud for 3 days); group II received IP injection of 4 g/kg/d of FE for 3 days; group III received IV injection of normal saline (3 mL/d for 3 days). Groups IV, V. and VI received IV injection of FE (4 g/kg/dose) over 15 minutes, at 4 hours, 24 hours, and 4 and 24 hours, respectively, prior to bacterial challenge. Radiolabeling of Bacteria The procedures used for radiolabeling of bacteria have been recently reported in detail” and will be briefly described: E coli (clinical isolate; Indiana University Hospital, Indianapolis, IN) was cultured to late exponential phase in a chemically defined medium containing 5 pXi/mL [“S] Na,SO, (New England Nuclear, Boston, Ma). “S-labeled bacteria were harvested and washed. Bacterial concentration was adjusted spectrophotometrically to approximately lo9 viable E coli per milliliter. Samples of the suspension were obtained for radioactivity determination prior to use.
Bacterial Clearance and Organ Localization For bacterial blood clearance and organ localization studies, the animals were sedated with an IM injection of ketamine cocktail and kept in a restraining cage. One milliliter of ?S-labeled viable E coli (lo9 bacteria/ml) was injected via the tail vein. For clearance, blood samples (50 kL) were collected using heparinized micropipettes into scintillation glass vials at 1,5, and 10 minutes following
From the Section of Pediatric Surgery, Department of Surgery, and the Department of Microbiology and Immunology, Indiana University School of Medicine, and the James Whitcomb Riley Hospital for Children, Indianapolis, IN. Presented at the Jens G. Rosenkrantz Resident Competition at the 42nd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Boston, Massachusetts, October 6-7, 1990. Address reprint requests to Jay L. Grosfeld, MD, Surgeon-in-Chief, J. W: Riley Hospital for Children, 702 Bamhill Dr (K-21) Indianapolis, IN 46202-5200. Copyright 0 1991 by W.B. Saunders Company 0022-3468/91/2608-0008$03.00!0 921
KATZ ET AL
922
bacterial challenge. At 10 minutes, the liver, spleen, kidney, and lung were immediately removed. Small tissue samples (50 to 100 mg) of the liver, spleen, kidney, and lungs as well as the blood samples were processed for liquid scintillation counting and expressed in disintegrations per minute (dpm). The final distribution of bacteria in the different organs was calculated from the input specific radioactivity (dpm/bacteria) and expressed as percentage of the injected bacteria per gram of tissue and per total organ. The experimental groups were studied concurrently with controls using the same bacterial concentration at 4,24, and 4 and 24 hours following administration of FE.
X OF INJECTED
60
LIVER
Radioactivity Determination Samples of cultured bacteria, blood, and tissue were dried and dissolved by treatment of 1.0 mL of 100% Soluene 350 (Packard Camberra Co, Downers Grove, IL) tissue solubilizer overnight at 50°C. The dissolved tissues were dried by 100% isopropyl alcohol and decolorized by 30% H,O, for 30 minutes and cooled to room temperature. Fifteen milliliters of Insta-Gel scintillation fluid (Packard Instrument Co, Downers Grove, IL) was added. Radioactivity was determined using a 2200 CA Tricarb analyzer (Packard Instrument Co, Downers Grove, IL) interfaced with an IBM microcomputer. Data were corrected for quench using a calibration curve determined by external standardization, and radioactivity was expressed as ‘5Sdpm.
Biochemistry Blood samples for serum cholesterol, free fatty acid (FFA), and triglycerides were obtained from 3 rats in each experimental group at the time of bacterial challenge.
Statistical Analysis Results were expressed as mean percent of injected bacteria f standard deviation. The differences in blood clearance and organ localization of bacteria were evaluated for validity by one-way analysis ofvariance(ANOVA) and the Student’s t test. RESULTS
There was no mortality following administration of IP and IV FE. The serum levels of cholesterol, FFA, and triglycerides are summarized in Table 1. There was no significant difference in these parameters in rats following IP injection compared with controls. Significant increase in blood levels of FFAs and triglycerides were found in rats at 4 (group IV) and 4 and 24 (group VI) hours following IV administration of FE.
Table 1. Serum Triglycerides. Free Fatty Acids, and Cholesterol in Flats Triglycerides Group
Control IP
Free Fatty Acids (kmol/L)
110 f 18
550 -c 70
65?
75 * 20
518 2 65
87 + 17 77 % 14
IV 4 h
800 f 95
2200 lr 350
IV24h
110 t 23
770 2 90
1350 f 260
2460 2 300
IV4and24h
Cholesterol
(mg/dL)
NOTE. n = 3 in each group. Abbreviations: IP, intraperitoneal; IV, intravenous.
MgldL)
72?
10
16
133 + 42
BACTERIA
60
SPLEEN
KIDNEY
LUNG
Fig 1. Comparative liver, spleen, kidney, and lung localization of IV injected radiolabeled E co/i in rats following IP injection of normal saline (n = 10) (D) and FE (a) 20% for 3 days. Values represent mean percent of injected bacteria (dpm/organ). Note a significant increase in lung localization of bacteria in FE rats.
Bacterial Clearance and Oqan Localization
IV injection of viable radiolabeled E coli was followed by immediate clearance of bacteria from the blood; 95% of the injected bacteria disappeared from the blood stream in the first 5 minutes. There was no significant change in the clearance rate of viable bacteria among the experimental groups. However, administration of FE did affect bacterial localization in the different organs. IP injection for 3 days results in an increase in lung localization of bacteria (3.7% + 1.5% v control 1.6% + 0.56%; P < .05) with no change in the ability of the liver to trap bacteria (Fig 1). IV administration of FE was followed by an increase in lung localization of bacteria in all the rats. Reduced bacterial trapping by the liver was found in rats at 24 hours (group V) and 4 and 24 hours (group VI) following IV administration of FE (Fig 2). DISCUSSION
The importance of aggressive nutritional support in improving host defenses and decreasing the risk of morbid infection has been clearly documented. Theoretically, FE being a major source of energy, is expected to further improve the resistance to infection. Paradoxically, it has been shown that “lipid overload” may result in various systemic complications. When the rate of lipid infusion exceeds the capacity of capillary endothelial lipoprotein lipase to metabolize lipid-emulsion triglycerides, the excess lipid may either coat, or be engulfed by, phagocytes and macrophages interfering with their normal function.4x9.10 Nugent” demonstrated that in vitro intralipid reduced phagocytic and pinocytic activities of mouse peritoneal macrophages. In contrast, adding FE to the parenteral nutrition regimen in cancer patients did not affect neutrophil chemotaxis and bacteriotidal activity.‘* Most of the data concerning the effect of FE on host immune response were obtained in in
INTRALIPID AND RES BACTERIAL CLEARANCE
% OF INJECTED
923
BACTERIA
* **
P-Eon5
P
