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Bacterial clearance in the intact and regenerating liver
Bacterial Clearance in the Intact and Regenerating Liver By Kirby Gross, Schmuel Katz, Stephen P. Dunn, Dolores Cikrit, Raoul Rosenthal, and Jay L. Grosfeld Indianapolis, Indiana 9 The Kupffer cells in the liver play an important role in reticuloendothelial system (RES) function by clearing particulate matter and bacteria from the blood stream. While hepatocyte regeneration and function have been extensively studied following partial hepatectomy, little information is available concerning RES function in the regenerating liver. This study investigates hepatic RES function by evaluating bacterial clearance (live E, coil) in the intact and regenerating liver. Thirty-four young male Sprague Dawley rats w e r e studied. T w e n t y - t w o animals u n d e r w e n t a standard 7 0 % partial hepatectomy using ligature technique and 12 had e sham operation. Both groups of rats received 109 organism of S 35 labeled E co/i, intravenously at 24 hours, 72 hours, 21/2 weeks, and 6 w e e k s postoperatively. Rats w e r e killed 10 minutes following injection and liver, lung, spleen, and kidney harvested, fixed, and radioactivity was determined using a scintillation spectrometer interfaced with a micro-computer counting the S 35 radiolabel. The total organ count of trapped bacteria in liver in partially hepatectomized rats was lower t h a n intact controls at 2 4 hours (22.0% v 4 6 . 4 % , P < .01 ), but was similar at 72 hours, 21/= weeks, and 6 weeks. Partial hepatectomy increased the amount of bacterial trapping in the lung at 24 hours (11.3% v 1.7%, P < .01) and 72 hours (10.1% v 1.7%, P < .05) and returned to normal at 21/= w e e k s and 6 weeks. Splenic activity was increased following hepatectomy at 21/2 weeks. Renal clearance was increased at 72 hours and 21/2 weeks. The amount of trapped bacteria per gram of liver was increased following partial hepatectomy at 24 hours (8,2% v 5.6%, P < .01) and 72 hours, (11.4% v 5.7%). Following partial hepatectomy, total organ hepatic bacterial clearance is decreased in the first 24 hours, and is associated with a concominant increased localization of live bacteria in lung. This suggests that Kupffer cell regeneration lags behind hepatocyte regeneration (which in the rat peaks at 24 hours). Bacterial clearance is extremely efficient at 72 hours and 2 weeks and then returns to normal at 6 weeks. Although the total organ clearance of bacteria was significantly less in partially hepatectomized rats, there was an increased clearance per gram of regenerating liver tissue. However, the total liver mass at 24 hours was inadequate for appropriate clearance. These observations suggest that early perioperative antibiotics may be useful in the clinical setting during the first 24 to 72 hours following partial hepatectomy. 9 1 9 8 5 by Grune & S t r a t t o n , Inc.
From the Section of Pediatric Surgery, the James Whitcomb Riley Hospital for Children, and the Department of Microbiology, Indiana University School of Medicine, Indianapolis. Presented at the 33rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Chicago, September 1~16, 1984. Address reprint requests to Jay L. Grosfeld, MD, Surgeonin-Chief, J.W. Riley Hospital for Children, 702 Barnhill Drive (K21), Indianapolis, IN 46223. 9 1985 by Grune & Stratton, Inc. 0022-3468/85/2004~9005503.00/0 320
INDEX W O R D S : Reticuloendothelial system; liver regeneration; hepatectomy.
HE KUPFFER CELLS of the liver play an important role in reticuloendothelial system (RES) function by clearing particulate matter and bacteria from the blood stream. Although hepatocyte regeneration and function has been extensively studied following partial hepatectomy, little information is available regarding RES function in the regenerating liver. With hepatic resection a standard part of current surgical therapy, it was considered important to study RES function in the regenerating liver. The purpose of this study was to investigate hepatic RES function by evaluating bacterial clearance (live E. coli) in both the intact and regenerating liver.
T
MATERIALS AND METHODS Thirty-four young male Sprague-Dawley rats weighing 150 to 175 grams were housed two per cage and fed with standard Wayne Lab-Block (Rat Chow 5001, Ralston Purina, St. Louis) and tap water ad libitum. Laparotomy was performed under ether anesthesia and clean technique. Twenty-two animals underwent a standard 70% partial hepatectomy using ligature technique and 12 animals had a sham operation. Animals were placed into groups to be studied at 24 hours, 72 hours, 2y2 weeks, and 6 weeks after operation.
Radiolabeling of Bacteria Escherichia coil (clinical isolate, Indiana University Hospital, Indianapolis) was cultured to late exponential phase at 39 ~ in a shaker bath in 10 mL of chemically-defined liquid medium containing 5 Ci/mL of (35S) Na2SO4 (New England Nuclear, Boston). 35S-labeled bacteria were harvested and washed three times by centrifugation (27,000 rpm for 1 minute in 0.9% sterile saline. Bacterial concentration was adjusted spectrophotometrically (Spectronic 20, Bausch and Lomb), to an absorbance of 540 mL that corresponded to approximately 109 viable E coli per mL as determined by a standard spread plate technique. Samples of the suspension were taken for radioactivity determination prior to utilizing the labeled bacteria in clearance and organ localization studies. Typically, the final specific activity of 35S was 106 to 107 disintegrations per minute per 109 viable bacteria. The experimental groups were studied concurrently with controls using the same bacterial suspension.
Organ Localization Studies For bacterial challenge and organ distribution determination, the animals were kept in a restraining animal cage. The tail was soaked in warm water (50 ~ and cleansed with ethanol 70%. One mL of 35S-labeled viable E. coil (109 bacteria/mL) was injected via a lateral tail vein using a ButterflyR infusion set (27 x 3/8, 8" tubing No. 4395) (Abbott Hospital Inc, Chicago). Journal of Pediatric Surgery, Vol 20, No 4 (August), 1985: pp 320-323
321
HEPATIC BACTERIAL CLEARANCE
Table 1. Liver Weight Following Standard Partial Hepatectomy Time After Operation
Weight
2 4 hours
2 . 5 8 + .71 gm
72 hours
7.51 _+ 1.O9 gm
2.5 weeks
10.25 _+ 1.56 gm
6 weeks
11.87 _+ 2 . 7 3 gm
100
]
L~0--
Determination
Samples of cultured bacteria in scintillation vials were dried and dissolved by treatment in 0.5 mL of 90% NC5 TM (Amersham Corp., Arlington Heights, Ill) tissue solubilizer for 30 minutes at 34 ~ prior to adding 10 mL of a toluene based scintillation fluid. Samples of excised tissues were dissolved by treatment of 1.0 mL of 100% NC5 T M at 50 ~ overnight. The tissues were decolorized by treatment with 0.5 mL of 30% H202 (Baker Analyzed Reagent, Phillipsburg, N J) for one hour at 50 ~ Samples were cooled to room temperature and 16 mL of scintillation fluid was added. Radioactivity was determined using a Packard Model 3255 scintillation spectrometer (Packard Instrument Co., Downers Grove, Ill) interfaced with an Apple II microcomputer. Data were corrected for quench using a calibration curve determined by external standardization, and radioactivity was expressed at 35S dpm.
PARTIAL HEPATECTOMY p<.01
60-
Ten minutes following injection of E coli, liver, spleen, kidneys and lungs were immediately removed and weighed. Small tissue samples (50 to 100 mg) were processed for liquid scintillation counting. The final distribution of bacteria in the different organs was calculated from the input specific activity (dpm/bacteria) and expressed as percentage of injected bacteria per gram of tissue and per total organ weight. Radioactivity
[•CONTROL
80
f
20--
SPLEEN
LIVER
KIDNEY
LUNG
Fig 1. Organ distribution of intravenously injected radiolabled viable E coil in controls and experimental animals 2 4 hours following partial hepatectomy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ. A significant decrease of liver trapping is observed with a concominant increase in lung uptake of bacteria.
RESULTS
Liver Weight to Body Weight Ratio The weight of the liver following partial hepatectomy increased rapidly. Regenerated liver weights were all significantly different from weights at 24hours posthepatectomy (Table 1). Organ Localization of Bacteria
Statistical Analysis Results were expressed as the mean _+ standard deviation. Data were analyzed using the paired students' t-test.
There was a significant increase in the localization of 35S radiolabeled live E coil per gram of liver tissue,
Table 2. Organ Localization of IV Injected Viable E Coil* Time After Operation
24 Hours Controls
Liver
Spleen
Kidney
Lung
5,60 _+ 1.16
3.92 _+ 1.79
.12 _+ .07
1.27 _+ ,67
8.20 + 1.50!"
6.08 +_ 3.25
.25 _+ .11
9.59 _+ 4.991"
5.67 + 2.19
3.09 _+ .58
.14 _+ .01
1.13 _+ .07
11.4 _+ 2.561"
3.42 + 1.53
.20 _+ .031"
7.12 + 3.001"
6.92 _+ 1.41
3.90 + .72
.47 _+ .03
2.36 _+ .34
10.68 _+ 4.11
1.95 _+ .26t
.41 _+ .041"
1.76 _+ .61
2.92 _+ .22
3.91 _+ 1.05
.03 _+ .01
.42 _+ .16
3.02 _+ 1.80
3.14 _+ 1.61
.08 _+ .05
.37 _+ .23
(n = 4) Partial Hepatectomy (n = 5) 72 Hours Controls (n = 2)
Partial hepatectomy (n = 6) 2.5 Weeks Controls (n = 4) Partial Hepatectomy (n = 5) 6 Weeks Controls (n = 2)
Partial Hepatectomy (n = 6)
*Values represent mean _+ SD expressed as percent of injected bacteria localized in 1 mg of tissue. tStatistically significant at P < .O1 v s h a m control.
322
GROSS ET AL 100 100-
r~CONTROL O
80-
/
8O
CONTROL
[ ~ I P A R T I A L HEPATECTOMY
[ 7 ~ P A R T I A L HEPATECTOMY 6O
p<.o5 ~
60-
p < . oos ~-
40
40. 20
20-
~r#t
LIVER
, ]E , . I . .
. -....'_.
SPLEEN
KIDNEY
LIVER LUNG
Fig 2. Location of radiolabled E coil in controls and animals following partial hepatectorny at 72 hours postoperatively. Values expressed as mean _+ SD of percent of injected bacteria per total organ, The liver and kidney in controls have less of the aSS radiolable.
at 24 hours and 72 hours posthepatectomy. This difference however, was not present at 2l/2 weeks and 6 weeks after partial hepatectomy. The lung showed a significant increase in trapped bacteria per gram of tissue at 24 hours and 72 hours after hepatectomy (Table 2). However, the total organ uptake of radiolabled E coli in the regenerating liver is significantly less than the control liver at 24 hours (Fig 1). The lung demonstrates an increased uptake of bacteria during early liver regeneration (first 24 hours). By 72 hours, however, the regenerated liver mass is adequate to normally trap radiolabeled E coli (Fig 2). Of interest is /It 100"
80-
II/
]
CONTROL
/I/ /i/
[]
PARTIAL HEPATECTOMY
~p<.001
// I// 60.
//;,
u,O.
// 20.
I// LIVER
= I
= v
SPLEEN
-
KIDNEY
n Z
v]~
,
LUNG
Fig 3. Distribution of 3BS labled E coil in controls and experimental animals 21/2 weeks following partial hepatectemy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ. The spleen in control animals had a significantly greater uptake of bacteria.
SPLEEN
KIDNEY
LUNG
Fig 4. There was no significant difference in localization of SSS E coli between controls and experimental animals at 6 weeks following partial hepatectomy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ.
the fact that the lung continues to demonstrate increased phagocytic function at 72 hours post hepatectomy. Splenic activity was increased at 21/z weeks and renal clearance was increased at 72 hours and 21/2 weeks post hepatectomy. By 21/2 and 6 weeks (Figs 3 and 4), the uptake of bacteria in both liver and lung is similar to control animals. DISCUSSION
Liver regeneration is characterized by a wave of proliferative activity that involves the parenchymal cells (hepatocytes) almost exclusively. 1 D N A synthesis usually begins at 15 to 18 hours and reaches a peak level at 24 to 26 hours. 2 Bile duct and supportive tissue cells enter D N A synthesis and mitosis much later; a maximal rate of D N A formation is not achieved in these tissues until 36 to 42 hours post hepatectomy. 2 This study suggests that Kupffer cell regeneration is delayed from 24 to 48 hours following partial hepatectomy. By 72 hours, bacterial clearance may exceed normal levels. Of interest is the fact that function of the reticuloendothelial tissue actually predates the maximal rate of D N A formation in non-hepatocyte tissue. At 24 hours, the regenerating liver demonstrated an increased clearance of radiolabeled E coli per gram of liver tissue. The total liver uptake of radiolabeled E coli however, was significantly depressed at 24 hours due to a decreased liver mass. The lung demonstrated increased uptake of bacteria. This was in response to an altered uptake of bacteria by the liver. This is consistent with other findings from our laboratory which demonstrated
HEPATIC BACTERIAL CLEARANCE
increased b a c t e r i a l localization in the lung in a n i m a l s with decreased hepatic R E S function associated with obstructive j a u n d i c e . 3 These observations suggest t h a t early p e r i o p e r a t i v e antibiotics m a y be useful in the clinical setting d u r i n g the first 24 to 72 hours following p a r t i a l h e p a t e c t o m y , at a time when a significant portion of the liver reticuloendothelial function is depressed.
323
REFERENCES
1. Grosfe|d JL, Weber TR, Baehner RL: Comparative toxicity of chemotherapy following partial hepatectomy. J Pediatr Surg 16:950-954, 1981 2. Bucher NCR, Swaffield MN, Ditroia JF: The influence of age upon the incorporation of thymidine 2 - 14C into DNA of regenerating rat liver. Cancer Res 24:509-512, 1964 3. Katz S, Grosfeld JL, Gross K: Impaired bacterial clearance and trapping in obstructive jaundice. Ann Surg 199:14-20, 1984
From the Section of Pediatric Surgery, the James Whitcomb Riley Hospital for Children, and the Department of Microbiology, Indiana University School of Medicine, Indianapolis. Presented at the 33rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Chicago, September 1~16, 1984. Address reprint requests to Jay L. Grosfeld, MD, Surgeonin-Chief, J.W. Riley Hospital for Children, 702 Barnhill Drive (K21), Indianapolis, IN 46223. 9 1985 by Grune & Stratton, Inc. 0022-3468/85/2004~9005503.00/0 320
INDEX W O R D S : Reticuloendothelial system; liver regeneration; hepatectomy.
HE KUPFFER CELLS of the liver play an important role in reticuloendothelial system (RES) function by clearing particulate matter and bacteria from the blood stream. Although hepatocyte regeneration and function has been extensively studied following partial hepatectomy, little information is available regarding RES function in the regenerating liver. With hepatic resection a standard part of current surgical therapy, it was considered important to study RES function in the regenerating liver. The purpose of this study was to investigate hepatic RES function by evaluating bacterial clearance (live E. coli) in both the intact and regenerating liver.
T
MATERIALS AND METHODS Thirty-four young male Sprague-Dawley rats weighing 150 to 175 grams were housed two per cage and fed with standard Wayne Lab-Block (Rat Chow 5001, Ralston Purina, St. Louis) and tap water ad libitum. Laparotomy was performed under ether anesthesia and clean technique. Twenty-two animals underwent a standard 70% partial hepatectomy using ligature technique and 12 animals had a sham operation. Animals were placed into groups to be studied at 24 hours, 72 hours, 2y2 weeks, and 6 weeks after operation.
Radiolabeling of Bacteria Escherichia coil (clinical isolate, Indiana University Hospital, Indianapolis) was cultured to late exponential phase at 39 ~ in a shaker bath in 10 mL of chemically-defined liquid medium containing 5 Ci/mL of (35S) Na2SO4 (New England Nuclear, Boston). 35S-labeled bacteria were harvested and washed three times by centrifugation (27,000 rpm for 1 minute in 0.9% sterile saline. Bacterial concentration was adjusted spectrophotometrically (Spectronic 20, Bausch and Lomb), to an absorbance of 540 mL that corresponded to approximately 109 viable E coli per mL as determined by a standard spread plate technique. Samples of the suspension were taken for radioactivity determination prior to utilizing the labeled bacteria in clearance and organ localization studies. Typically, the final specific activity of 35S was 106 to 107 disintegrations per minute per 109 viable bacteria. The experimental groups were studied concurrently with controls using the same bacterial suspension.
Organ Localization Studies For bacterial challenge and organ distribution determination, the animals were kept in a restraining animal cage. The tail was soaked in warm water (50 ~ and cleansed with ethanol 70%. One mL of 35S-labeled viable E. coil (109 bacteria/mL) was injected via a lateral tail vein using a ButterflyR infusion set (27 x 3/8, 8" tubing No. 4395) (Abbott Hospital Inc, Chicago). Journal of Pediatric Surgery, Vol 20, No 4 (August), 1985: pp 320-323
321
HEPATIC BACTERIAL CLEARANCE
Table 1. Liver Weight Following Standard Partial Hepatectomy Time After Operation
Weight
2 4 hours
2 . 5 8 + .71 gm
72 hours
7.51 _+ 1.O9 gm
2.5 weeks
10.25 _+ 1.56 gm
6 weeks
11.87 _+ 2 . 7 3 gm
100
]
L~0--
Determination
Samples of cultured bacteria in scintillation vials were dried and dissolved by treatment in 0.5 mL of 90% NC5 TM (Amersham Corp., Arlington Heights, Ill) tissue solubilizer for 30 minutes at 34 ~ prior to adding 10 mL of a toluene based scintillation fluid. Samples of excised tissues were dissolved by treatment of 1.0 mL of 100% NC5 T M at 50 ~ overnight. The tissues were decolorized by treatment with 0.5 mL of 30% H202 (Baker Analyzed Reagent, Phillipsburg, N J) for one hour at 50 ~ Samples were cooled to room temperature and 16 mL of scintillation fluid was added. Radioactivity was determined using a Packard Model 3255 scintillation spectrometer (Packard Instrument Co., Downers Grove, Ill) interfaced with an Apple II microcomputer. Data were corrected for quench using a calibration curve determined by external standardization, and radioactivity was expressed at 35S dpm.
PARTIAL HEPATECTOMY p<.01
60-
Ten minutes following injection of E coli, liver, spleen, kidneys and lungs were immediately removed and weighed. Small tissue samples (50 to 100 mg) were processed for liquid scintillation counting. The final distribution of bacteria in the different organs was calculated from the input specific activity (dpm/bacteria) and expressed as percentage of injected bacteria per gram of tissue and per total organ weight. Radioactivity
[•CONTROL
80
f
20--
SPLEEN
LIVER
KIDNEY
LUNG
Fig 1. Organ distribution of intravenously injected radiolabled viable E coil in controls and experimental animals 2 4 hours following partial hepatectomy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ. A significant decrease of liver trapping is observed with a concominant increase in lung uptake of bacteria.
RESULTS
Liver Weight to Body Weight Ratio The weight of the liver following partial hepatectomy increased rapidly. Regenerated liver weights were all significantly different from weights at 24hours posthepatectomy (Table 1). Organ Localization of Bacteria
Statistical Analysis Results were expressed as the mean _+ standard deviation. Data were analyzed using the paired students' t-test.
There was a significant increase in the localization of 35S radiolabeled live E coil per gram of liver tissue,
Table 2. Organ Localization of IV Injected Viable E Coil* Time After Operation
24 Hours Controls
Liver
Spleen
Kidney
Lung
5,60 _+ 1.16
3.92 _+ 1.79
.12 _+ .07
1.27 _+ ,67
8.20 + 1.50!"
6.08 +_ 3.25
.25 _+ .11
9.59 _+ 4.991"
5.67 + 2.19
3.09 _+ .58
.14 _+ .01
1.13 _+ .07
11.4 _+ 2.561"
3.42 + 1.53
.20 _+ .031"
7.12 + 3.001"
6.92 _+ 1.41
3.90 + .72
.47 _+ .03
2.36 _+ .34
10.68 _+ 4.11
1.95 _+ .26t
.41 _+ .041"
1.76 _+ .61
2.92 _+ .22
3.91 _+ 1.05
.03 _+ .01
.42 _+ .16
3.02 _+ 1.80
3.14 _+ 1.61
.08 _+ .05
.37 _+ .23
(n = 4) Partial Hepatectomy (n = 5) 72 Hours Controls (n = 2)
Partial hepatectomy (n = 6) 2.5 Weeks Controls (n = 4) Partial Hepatectomy (n = 5) 6 Weeks Controls (n = 2)
Partial Hepatectomy (n = 6)
*Values represent mean _+ SD expressed as percent of injected bacteria localized in 1 mg of tissue. tStatistically significant at P < .O1 v s h a m control.
322
GROSS ET AL 100 100-
r~CONTROL O
80-
/
8O
CONTROL
[ ~ I P A R T I A L HEPATECTOMY
[ 7 ~ P A R T I A L HEPATECTOMY 6O
p<.o5 ~
60-
p < . oos ~-
40
40. 20
20-
~r#t
LIVER
, ]E , . I . .
. -....'_.
SPLEEN
KIDNEY
LIVER LUNG
Fig 2. Location of radiolabled E coil in controls and animals following partial hepatectorny at 72 hours postoperatively. Values expressed as mean _+ SD of percent of injected bacteria per total organ, The liver and kidney in controls have less of the aSS radiolable.
at 24 hours and 72 hours posthepatectomy. This difference however, was not present at 2l/2 weeks and 6 weeks after partial hepatectomy. The lung showed a significant increase in trapped bacteria per gram of tissue at 24 hours and 72 hours after hepatectomy (Table 2). However, the total organ uptake of radiolabled E coli in the regenerating liver is significantly less than the control liver at 24 hours (Fig 1). The lung demonstrates an increased uptake of bacteria during early liver regeneration (first 24 hours). By 72 hours, however, the regenerated liver mass is adequate to normally trap radiolabeled E coli (Fig 2). Of interest is /It 100"
80-
II/
]
CONTROL
/I/ /i/
[]
PARTIAL HEPATECTOMY
~p<.001
// I// 60.
//;,
u,O.
// 20.
I// LIVER
= I
= v
SPLEEN
-
KIDNEY
n Z
v]~
,
LUNG
Fig 3. Distribution of 3BS labled E coil in controls and experimental animals 21/2 weeks following partial hepatectemy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ. The spleen in control animals had a significantly greater uptake of bacteria.
SPLEEN
KIDNEY
LUNG
Fig 4. There was no significant difference in localization of SSS E coli between controls and experimental animals at 6 weeks following partial hepatectomy. Values represent mean _+ SD expressed as percent of injected bacteria per total organ.
the fact that the lung continues to demonstrate increased phagocytic function at 72 hours post hepatectomy. Splenic activity was increased at 21/z weeks and renal clearance was increased at 72 hours and 21/2 weeks post hepatectomy. By 21/2 and 6 weeks (Figs 3 and 4), the uptake of bacteria in both liver and lung is similar to control animals. DISCUSSION
Liver regeneration is characterized by a wave of proliferative activity that involves the parenchymal cells (hepatocytes) almost exclusively. 1 D N A synthesis usually begins at 15 to 18 hours and reaches a peak level at 24 to 26 hours. 2 Bile duct and supportive tissue cells enter D N A synthesis and mitosis much later; a maximal rate of D N A formation is not achieved in these tissues until 36 to 42 hours post hepatectomy. 2 This study suggests that Kupffer cell regeneration is delayed from 24 to 48 hours following partial hepatectomy. By 72 hours, bacterial clearance may exceed normal levels. Of interest is the fact that function of the reticuloendothelial tissue actually predates the maximal rate of D N A formation in non-hepatocyte tissue. At 24 hours, the regenerating liver demonstrated an increased clearance of radiolabeled E coli per gram of liver tissue. The total liver uptake of radiolabeled E coli however, was significantly depressed at 24 hours due to a decreased liver mass. The lung demonstrated increased uptake of bacteria. This was in response to an altered uptake of bacteria by the liver. This is consistent with other findings from our laboratory which demonstrated
HEPATIC BACTERIAL CLEARANCE
increased b a c t e r i a l localization in the lung in a n i m a l s with decreased hepatic R E S function associated with obstructive j a u n d i c e . 3 These observations suggest t h a t early p e r i o p e r a t i v e antibiotics m a y be useful in the clinical setting d u r i n g the first 24 to 72 hours following p a r t i a l h e p a t e c t o m y , at a time when a significant portion of the liver reticuloendothelial function is depressed.
323
REFERENCES
1. Grosfe|d JL, Weber TR, Baehner RL: Comparative toxicity of chemotherapy following partial hepatectomy. J Pediatr Surg 16:950-954, 1981 2. Bucher NCR, Swaffield MN, Ditroia JF: The influence of age upon the incorporation of thymidine 2 - 14C into DNA of regenerating rat liver. Cancer Res 24:509-512, 1964 3. Katz S, Grosfeld JL, Gross K: Impaired bacterial clearance and trapping in obstructive jaundice. Ann Surg 199:14-20, 1984