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Jaundice Associated with Peritoneal Absorption of Bilirubin
GASTHOENTEHOLO(;y
Vol. GH, No. ;,
Copyright © 1970 by The \Villiams & Wilkins Co,
11rintecl in l!. .0...,'. A.
CASE REPORTS
JAUNDICE ASSOCIATED WITH PERITONEAL ABSORPTION OF BILIRUBIN P.
STATIIAKOS,
G.
M.
M.D., R PH.D.
FANSKA, B.S.,
,J. V.
CAIUIONE,
M.D.,
AND
GHODSKY,
Division of Gastroenterology, Department of Medicine, The Permanente Medical Group, Kaiser Foundation Hospital, Oahland, and the Department of Biochemistry and Biophysics, the Department of Medicine, and the Metabolic Research Unit of the Department of Medicine, University of California School of Medicine, San Francisco, California
Jaundice was observed in a patient whose common bile duct was completely severed for 26 days. Absorption of the extravasated bilirubin and varying diversion of biliary flow into the peritoneal cavity contributed to the icterus. Because peritoneal absorption of bilirubin had not been previously defined, studies were conducted in the rat. In bile fistula and lymph fistula rats, absorption was determined by the intraperitoneal injection of aH-bilirubin or by application of the isotopic pigment on peritoneal membranes. Unconjugated bilirubin and bilirubin conjugates were both rapidly absorbed. Studies in the Gunn rat indicated that bilirubin conjugates could pass the peritoneal membranes without being converted to unconjugated bilirubin. Absorption by way of the thoracic lymph accounted for about 11 'X, of the peritoneal absorption; the remainder was presumably absorbed by way of the diaphragmatic lymph and by diffusion through membranes of the parietal and visceral peritoneum. In rats in which the common bile duct was transected, overgrowth of the duct by adhesions stopped biliary flow into the peritoneal cavity within 24 hr. Alternatively, a polyethylene cannula was inserted into the bile duct. With bile freely effusing into the peritoneal cavity for periods of 19 to 48 hr, serum bilirubin was elevated. Most of the hyperbilirubinemia was found to be due to the rapid cyclic absorption of bilirubin from the peritoneal space and back into it sequentially by way of the blood, liver, and bile duct. Jaundice in the patient is discussed in view of the findings in the rat. A number of conditions are known to result in the excretion of bile into the peri-
toneal cavity: idiopathic infantile bile peritonitis, spontaneous idiopathic bile peritonitis, and blunt trauma to the abdo-
Received June 20, 1969. Accepted June 2, 1970. Address requests for reprints to: Dr. G. M. Grodsky, Metabolic Research Unit, University of Califor· Group, Kaiser Foundation Hospital, Oakland, Cali· nia School of Medicine, San Francisco, fornia, for referring the case for further study; H. California 94122. Stathakos for technical assistance; Barbara Haas, This investigation was supported by United Norma Brust, and K. Nemechek, of the University States Public Health Service Grant A-6115 and by of California Medical Center, for technical assist· Kaiser Foundation Hospital Grant 672-0 (66). ance; and Dr. Rudi Schmid, University of California The authors thank Dr. Harris H. Holboe of the Medical Center, for supplying Gunn rats used in this Department of Surgery, The Permanente Medical study and for review of the manuscript. 769
770
CASE REPORTS
men. Complete severance of the common bile duct resulting from blunt trauma to the abdomen, a rare event, is uniformly fatal without surgical intervention. 1- 3 In such a patient, hyperbilirubinemia was observed. There was also high concentration of bilirubin in the ascitic fluid. The absorption and disposition of this bilirubin had not been previously defined. In view of the possibility that the jaundice could have arisen from the absorption of bilirubin from the ascitic fluid, studies were conducted in the rat.
Case Report In a 19-year-old male, the common bile duct was completely severed by blunt trauma incurred in an automobile accident on day 1. By day 10 his abdomen was slightly distended, and serum bilirubin was elevated (fig. 1) . On days 13 to 16, serum bilirubin rapidly rose. On day 16, abdominal paracentesis yielded 8 liters of jaundiced fluid. The following day, serum bilirubin was lowered, presumably a result of the abdominal tap. No additional trauma to the patient was reported on day 16. Serum bilirubin remained essentially unchanged at the ' lower level until day 27, when the completely transected bile duct was transplanted to the duodenum; adhesions were seen between the liver and the abdominal wall. Mter the surgical procedure, serum bilirubin became normal and the patient returned to good health. During the 26 days before operation, alkaline phosphatase was 29 to 44 King-Armstrong
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units (days 15 to 24) ; serum glutamic oxaloacetic transaminase was 100 units (day 19) ; Bromsulphalein (BSP) retention was 16% (day 24), and about 7 g of bilirubin were excreted into the peritoneal cavity.•
Materials and Methods Labeled bilirubin in donor bile was injected intraperitoneally or applied on selected sites of visceral and parietal peritoneum in bile fistula and lymph fistula male Long-Evans rats and homozygous (jj) , jaundiced Gunn rats.5 Absorption of labeled pigment was determined by the subsequent excretion of radioactivity in bile and lymph or by assessment of isotope content of serum and liver. In rats with surgically transected bile duct, the relationship of jaundice to the peritoneal absorption of bilirubin was investigated. Counting and crystallizing tritiated bilirubin. Bilirubin isotope was counted in a Packard Tri-Carb liquid scintillation photospectrometer . Lymph (60 "liters), serum (20 "liters) , bile (20 "liters), or homogenized liver (25 mg) was diluted with 0.25 ml of water and freezedried. The dry sample was dissolved in 1.5 ml of a molar solution of Hyamine in methanol in the presence of 90 "liters of 30% hydrogen peroxide and was diluted with 15 ml of scintillator solution. 6 All samples were corrected for quenching by measuring the recovery of "Hglycine added to a duplicate sample. Aliquots of bile containing "H-bilirubin were assayed for degraded isotope by the bilirubin crystallization method of Ostrow eta!. 7 Chemical determination of bilirubin in ser-
e 0
0
0 Days after automobile accident FIG .
1. Serum bilirubin levels in the patient.
Novemb er 1970
C ASE REPORTS
um, bile, and peritoneal fluids. Direct and total bilirubin was determined by the diazo method of Malloy and Evelyn," except that the sulfanilic acid concentration of the reagent was increased 2-fold. Diazo standards were prepared with certified reagent bilirubin (Fisher Scientific Company). Serum was diluted 1:20 with water before reagent was added . Bile and peritoneal fluid were diluted with water so that the amount of bilirubin that reacted with 1 ml of diazo reagent was between 3 and 25 11 g. The maximum optical density for the determination of total bilirubin was obtained from photometric readings made at 3-min intervals. Under these conditions, photometric absorption was linear with bilirubin concentration. Collection of bile. Bile was collected in Wintrobe sedimentation tubes which had been covered with aluminum foil to exclude light and placed in an ice bath; subsequently, bile was frozen until just before use. Preparation of donor bile containing bilirubin conjugates . For injections or infusions in the rats, conjugated bilirubin was obtained from bile fistula donor rats given intravenous injections of 0 _1 M Na2C02 solution of tritiated bilirubin 9 or bilirubin (Eastman) . Injection of 150 11g of "H-bilirubin (specific activity 23 11 c per mg) , repeated hourly, resulted in biliary excretion of approximately 150 Jlg of total bilirubin per ml of bile (specific activity 15 11 c per ml) . A mean of 95% of the radioactivity in the bile was crystallizable "H-bilirubin. Injections of 1 mg of Eastman bilirubin per 100 g body weight at 20-min intervals gave rise to excretion of 8,000 t o 10,000 11g of unlabeled bilirubin per ml of bile . Labeled and unlabeled bile were usually combined and diluted with 0.9'/(, saline. Injectates and infusates contained approximately 1 ml of bile and 1 ml of saline . For paintings on small areas of parietal peritoneum, labeled bile of high specific activity was prepared by injecting 900 11g of "H-bilirubin hourly. This technique has been used for the preparation of labeled bilirubin conjugates in other studies, 10 12 even though the nature of the conjugates has not been established. Normally, most of the bilirubin excreted in bile is bilirubin diglucuronide. 1" However, significant excretion of polar complexes of unconjugated bilirubin with the diglucuronide, 14 or possibly as bilirubin monoglucuronide, can also occur. 15 In ancillary studies, we investigated these biliary products after intravenous injection of 1 mg of labeled bilirubin per 100 g of body weight into normal rats. By the modified diazo technique above, direct reacting bilirubin was 99 to 100% of the total. Paper chromatography
771
of the undiazotized pigments in n-propanol, nbutanol, and wa ter (3: 4: 3) at 4 C and in nitrogen atmosphere 1 ,; gave two major bands. More than 50'/;, of the total pigment was in a band correspondin g in mobility and glucuronide content to bilirubin diglucuronide. Most of the remainder was distributed into a less polar band containing variably less than 2 moles of glucuronic acid per mole of bilirubin, but with mobility different from lipophilic unconju gated bilirubin, the latter comprising less than 4% of the total pigment. In the present study, these normal biliary excretion products of bilirubin are referred to as bilirubin conjugates. Their absorption was often tested in the Gunn rat to establish whether absorbed tritiated pigment was or was not unconjugated bilirubin. Absorbed, unconjugated "H-bilirubin in the circulation of the Gunn rat would not be secreted by the liver and appear in the bile in amounts comparable to absorbed conjugated forms." Preparation of unconjugated bilirubin solutions for intraperitoneal injection in the rat. This was mad e by dissolving bilirubin and "Hbilirubin in 0.1 M Na2CO" to final concentration of 2 mg per mi. The pH was adjusted to 9.0 with 0.5 M HCl, and an equal volume of normal rat bile was added. Further dilution when required was made with 0.9'/(, saline . Biliary excretion of intraperitoneally injected "H-bilirubin conjugates and unconjugated bilirubin. After 1-cm midline incision in normal and Gunn rats, PE 50 polyethylene tubing (Clay Adams, Division of Beckton, Dickinson and Company) was introduced into the common bile duct. With the distal end of the cannula exterior to the rat, peritoneum, muscle, and skin were each sutured. After injection of donor bile containing labeled bilirubin conjugates or unconjugated bilirubin, recipient bile was collected at 1-hr intervals for 2 hr. Each bile was assayed for crystallizable isotope content. Excretion of intraperitoneally injected "Hbilirubin conjugates in thoracic lymph and bile. Normal rats received high fat diet for 24 hr before the thoracic duct was cannulated as previously des cribed by Bollman et al. 1" Each animal was placed in a Bollman restraining cage where it was fed stock rat diet and watered with 0.9 % NaCl ad libitum. The following day, each rat was removed from the cage and the common bile duct was cannulated. After injection of donor bile containing labeled bilirubin conjugates, recipient lymph and bile were collected at hourly intervals for 2 hr. Tritium in lymph and bile was counted, and "H-
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CASE REPORTS
bilirubin in bile was crystallized. Since cannulation of the thoracic duct was above the cisterna chyli, virtually all lymphatic drainage from the peritoneal cavity, except that by way of the diaphragmatic lymph, was collected. 19 ' 20 Absorption of 3 H-bilirubin conjugates by the visceral and parietal peritoneum . Absorption by the visceral peritoneum was assessed in normal rats. The common bile duct was cannulated. A midline incision was lengthened to allow extrusion of the intestine. The rat was positioned prone over a small beaker containing 2000 11 g of bilirubin conjugates (labeled with 2 11c of tritium) in 1 ml of bile diluted with 4 ml of saline. The ileum, cecum, and 1 em of colon were bathed. Bile was collected for 2 hr in the dark. Absorption by the abdominal wall (parietal peritoneum, drained by thoracic lymph) was demonstrated in normal and in Gunn rats. The bile duct was cannulated and the midline incision was extended from the xiphoid process to the pubis. From the ends of the incision, diagonal cuts were made which produced bilateral flaps of abdominal wall. The flaps were pulled to the sides of the rat by means of hemostats. A clamp composed of several thicknesses of aluminum foil was used to elevate the viscera above the flaps . In a darkened room, bile containing bilirubin conjugates was rolled onto the flaps with a cotton-tipped applicator at 7min intervals, over a period of 2 hr. Bile was collected during this period. The surface area of the parietal peritoneum was measured from imprints of the surfaces which were made on graph paper. Absorption by the diaphragm (parietal peritoneum, drained by diaphragmatic lymph) was shown in normal rats. The bile duct was cannulated and abdominal flaps were constructed as described above. Bile containing bilirubin conjugates was rolled onto the ventral diaphragm at intervals of 7 min, with the rat inclined 15°, head down. The quantity of bilirubin applied on the diaphragm or abdominal wall was determined from the difference between bilirubin originally assayed in the solution from which application was made and the sum of bilirubin (a) remaining in the solution, (b) on the applicator, and (c) rinsed from the topical site. The bile of all rats was assayed for isotope content and "H-bilirubin was crystallized from it. Relationship of jaundice to the peritoneal absorption of bilirubin in rats effusing bile into the peritoneal cavity. In 6 normal rats, the abdomen was opened, the common duct was
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transected at 1 em from its proximal end, and the peritoneum, muscle, and skin were each sutured. Mter 24 hr, exploratory laparotomy was performed to ascertain the reasons for the failure of fluid to accumulate in the peritoneal cavity, and the rats were killed. In the remainder of the rats, biliary effusion was prolonged by cannulating the bile duct with PE 50 polyethylene tubing. The cannula was clipped to 1cm length, and the distal tip was placed in the peritoneal cavity. The incision was sutured as above. The a nimals were allowed routine diet and water ad libitum without supportive therapy for periods up to 48 hr. Liver function and bile duct patency were assessed in 6 rats in which effusion was prolonged. Bromsulphalein, 5 mg per kg preoperative body weight, was injected intravenously while the cannula remained in the peritoneal cavity, or after midline incision was reopened to permit extension of the cannula to the exterior of the rat by forcing it into tightly fitting PE 90 tubing. The dye was assayed in bile or peritoneal f1uid by the method of Gaebler" 45 min after injection (in control rats, the common bile duct was cannulated; bile flow was directed either intraperitoneally or outside of the body, and BSP was injected without delay). Serum volume was determined from dilution of intravenously injected "11 1 human serum albumin. 22 Peritoneal absorption was assessed in 6 rats effusing bile into the peritoneal cavity by the appearance of label in serum and liver, after bile containing 3 H-bilirubin conjugates was injected intraperitoneally. Ten minutes after injection, the rats were decapitated and tritium content of serum and liver was determined. During this short period, most absorbed pigment can be accounted for in the serum and liver, since secretion into the bile does not have time to occur 23 (in an ancillary study, no surgery was performed before "H-bilirubin conjugates were injected). Cycling of endogenous bilirubin from and back into the peritoneal cavity was estimated in 9 rats effusing bile into the peritoneal cavity. The midline incision was reopened, and the cannula was extended outside the body. Bile was collected for 10 min, and each rat was decapitated. The rate of excretion of endogenous bilirubin in the bile and the bilirubin content of serum and peritoneal fluid were determined by the diazo reaction. Intrav enous infusion of "H-bilirubin conjugates in bile fistula rats to achieve hyperbilirubinemia comparable to that in rats effusing bile into the peritoneal cavity. Bile containing
November 1970
CASE REPORTS
labeled bilirubin conjugates in 6 different concentrations was infused into the inferior vena cava for 90 min . Serum and bile were assayed for tritium content at 15-min intervals during infusion. The infusion technique has been pre24 viously described.
Results Biliary excretion of intraperitoneally injected 3 H-bilirubin conjugates and unconjugated :1H-bilirubin. Rapid excretion of crystallizable isotope is shown in table 1. After injection of either 3 H-bilirubin conjugates or :1H-unconjugated bilirubin in normal rats, about 20% of the dose was recovered in bile by 2 hr; the rate of excretion was slightly higher during the second hour. 3 H-bilirubin conjugates were excreted in the bile of Gunn rats and normal rats at an equal rate. A mean of 94% of the isotope in the bile of the rats given injections of bilirubin conjugates was crystallizable. This indicated molecular integrity of excreted aH-bilirubin. When unconjugated "H-bilirubin was injected, a
mean of 89 % of the isotope appearing in bile was crystallizable. The mean bile volume excreted by the normal rats of the group was 0.93 ml per hr; the Gunn rats excreted 0.57 ml per hr. Excretion of intraperitoneally injected aH-bilirubin conjugates in thoracic lymph and bile. Rate of excretion in lymph and bile is shown in table 2. During the first 2 hr, label excreted in lymph and bile was 91 % of that excreted by rats with bile fistula only (table 1). Ninety-three per cent of the isotope was crystallizable from bile in this experiment. Approximately 11 (X, of the radioactivity excreted was through the lymphatic system. In contrast to bile, this pathway became less important during the second hour. After intraperitoneal injection, mean volumes of lymph and bile were 1.5 and 0.7 ml per hr, respectively. Absorption of aH-bilirubin conjugates by the visceral and parietal peritoneum. Rapid absorption by the visceral peritoneum when bathed in a solution of bilirubin conjugates was indicated as follows:
Biliary excretion of intraperitoneally injected "H-bilirubin conJulfates and unconjulfaled "Hbilirubin ( CB and UCB) in the rat"
1.
TABLE
773
1
No. of
Body
rats
weight
:
1
H -Biliruhin injected into peritoneal cnvity
i-I-Biliruhin excreted in hilc
Dur ing the lin;t hour nflcr injection
~ ·;. ofdww
I!
Normal 3 3 Gunn 3
397 ± 5 333 ± 17
CB (in bile) UCB (added to bile)
7.7 ± 0.6 8.3 ± 0.5
9.8 ± 0.4 10.6 ± 0.3
207 ± 2
CB
7.4 ± 0.5
9.7 ± 0.3
' Mean values (±sE) are shown. Quantity of bilirubin injected in all rats was 2000 of tritium. Volume injected was 2.0 mi. TABLE
2.
1 :
rats
Body weight
g
3
~g.
labeled with 2.0
~c
Excretion of intraperitoneally injected "H-bilirubin conjugates in thoracic lymph and bile of the rat' Qunntityof
No . of
During the second hour after injcclion
342 ± 14
1 :
H-hilirubin
injected into peritoneal cav ity
2~0
I
In bile
2.0
I
During the
During the first hour after injection
During the second hou r nftcr injection
nftcr injection
1.1 ± 0.5
0.6 ± 0.2
6.1 ± 0.8
first hour ~·;.
ml
pC
1 2.0
H-Bilimbin excreted
In lymph
During the second hour after injection
of dose
'f
8.2 ± 0.9
'Mean values (±sE) are shown. A cannula was placed in the abdominal thoracic duct and another in the common bile duct of each rat before injection.
774
CASE REPORTS
mean biliary excretion of crystallizable H-bilirubin absorbed by the ileum and cecum was 1.5% of the dose in the first hour and 1.6% in the second hour. These represent minimal values, since the surfaces bathed were visually estimated to be one-half those of the entire visceral peritoneum. A mean of 94% of the radioactivity excreted in bile was crystallizable 3 H-bilirubin. Rapid absorption of 3H-bilirubin conjugates by the parietal peritoneum in normal and Gunn rats is shown in table 3. In the Gunn rats, bilirubin conjugates were also rapidly absorbed by the abdominal wall and excreted in the bile. A mean of 87% of the radioactivity excreted in bile was crystallizable 3 H-bilirubin. The relationship of jaundice to the peritoneal absorption of bilirubin in rats effusing bile into the peritoneal cavity. In all of the rats in which a cannula was not placed in the bile duct, biliary flow into the peritoneal cavity ceased within 24 hr. Complete overgrowth of the duct by adhesions was seen. Results in the remaining
rats of this group were obtained by inserting a cannula into the severed bile duct. In a series of rats with 48-hr of effusion of bile into the peritoneal cavity, the percentage of intravenous dose of BSP found in ascitic fluid after 45 min was 68 ± 3 (n = 6); when bile flow was exteriorized, 84 ± 1% (n = 6) of the dose appeared in the bile. In either case, the mean value was 91 % of that found in control rats. In all rats having exteriorized bile cannula, BSP was not detected in ascitic fluid . These results indicated patency of the bile duct and minimal liver damage in rats with 48 hr of effusion of bile into the peritoneal cavity. Serum volume was 3.04 ± 0.08 ml per 100 g of preoperative body weight (n = 10), approximately 91 % of that in normals; hematocrit was 118% of normal. Large amounts of endogenous bilirubin were found in the peritoneal cavity by 48 hr (table 4). The animals had mild hyperbilirubinemia of 0.9 mg per 100 ml. Mean bilirubin concentration in peritoneal fluid was 12.8 times that in serum. The rate of absorption of a trace intraperitoneal dose
3
TABLE
No. of rats
Biliary excretion of 'H-bilirub in conjugates topically painted on the parietal peritoneum"
3.
aH·Bilirubin topically pain ted on parietal peritoneum
Body weight
During the Quantity
400 ± 17 390 ± 5
Gunn 3
207 ± 7
Abdominal wall Ventral diaphragm Abdominal wall
During the seco nd hour after injection
first hour
after injection
em '
li
6
:'H-Bi liruhin excreted in bile
Locus Total a rea painted
Normal 6
Vol . 59, No.5
~;, of
"c
1'1!
do.'ie
14
3050 ± 182
3. 0 ± 0. 2
4.7 ± 1.4
8.3 ± 0.5
4
3200 ± 278
3.2 ± 0.3
9. 1 ± 1.1
9.6 ± 1.3
10
2100 ± 200
2.1 ± 0.2
4.6 ± 1.5
8 .2 ± 1.4
" Mean valu es (±SE) are shown . The mea n surface area of the abdominal wall was 8.2 times the total area painted.
TABLE
4.
Distribution of endogenous bilirubin in rats in which the common bile duct was severed for 48 hr" Body weight
No. of rats
N ormal
l
Peritoneal fluid
370 ± 25
j
Volume
Total bilirubin
48 hr
Volume ml
,g
ml
,g
380 ± 27
25± 7
2980 ± 644
11.3 ± 0.3
105 ± 13
g
10
Serum
T ota l bilirubin
" Mean values (± sE) are shown. Normal serum contai ns less than 10 11g of total bilirubin.
November 1970
CASE REPORTS
775
3
of H-bilirubin conjugates was about equal to or greater than that in normal unoperated rats (table 5). Rates of cycling of endogenous bilirubin after 19 to 48 hr of bile effusion into the peritoneal cavity are shown in table 6. Excretion rate of bilirubin during the first 10 min after exteriorizing the bile duct minus normal bilirubin output in bile (normal bile fistula rats, n = 9, body weight = 371 ± 35 g; excretion = 9.0 ± 1.0 11 g bilirubin per 10 min) was taken as an estimate of cycling rate of bilirubin from the peritoneal space, through blood, liver, and bile, which was occurring immediately before bile duct translocation. Excretion rate during this time was about 15 times normal. Intravenous infusion of :1H-bi lirubin conjugates in bile fistula rats to achieve hyperbilirubinemia comparable to that in rats effusing bile into the peritoneal cavity. Regardless of the dose infused,. comparatively steady concentrations of 3 H-biTABLE
5.
lirubin serum (and bile) were reached before 90 min. In each of 6 rats, serum bilirubin content at 90 min was plotted on a graph against the infusion rate, together with serum bilirubin content and estimated cycling rate of each of 9 rats (summarily shown in table 6) effusing bile into the peritoneal cavity 19 to 48 hr. The result is shown in figure 2. Although the state of the rats in these two groups was quite different, a closely parallel linear relationship was obtained on the graph. Thus, most hyperbilirubinemia in rats in which the bile duct was severed appeared to be due to absorptive cycling of endogenous bilirubin. Discussion Comprehensive reviewsir.. il; indicate that many molecules rapidly pass through the peritoneal membranes in man and animals without distinction as to lipid solubility and molecular charge. Thus,
Rates of absorption of intraperitoneally injected "H-bilirubin conju~:ates in normal rats and rats effu~ing bile into the peritoneal cavity for 48 hr~" Normal body
No. of rats
weight
Qunntity of :t H-hiliruhin conjugates injected into pcritoncnl cnvity
1
Appenl"nnce of ' H-hilirubin
In serum
Serum
+
liver
I'll
pC
ml
369 ± 12
150
2.0
2.0
1.5 ± 0.1
2.5 ± 0.2
4.0 ± 0.3
365 ± 24
150
2.0
2.0
2.3 ± 0. 3
2.9 ± 0.4
5.2 ± 0.7
~
Normal, without surgery 6 Effusing bile for 48 hr 6
In liver
Hate of nhsorpti
.'j, of dose/10 min
1
' Mean values (±sE) are shown. TABLE
6.
Rate of cycling of endogenous bilirubin in rats effusing bile into the peritoneal cavity for 19 to 48 hr" Distribution of bilirubin after bile flow direc ted cxtraperitoncally during last 10 min of final hour
No. of rats
9
Normal body weight
In peritoneal fluid
II
I'll
370 ± 35
2320 ± 1182
Excreted in
In serum
Cyclinf.! of bilirubin
bile
~
w:/10 min
76 ± 31
146 ± 34
..
~«110
min/rat
137 ± 32
..
' Mean values (±sE) are shown . Bile duct transected 33 ± 10 hr. Bil1ary excretwn rate of b1hrubm while the bile duct was external to the rat minus bilirubin output in normal bile fistula rats (n = . 9, body weight = 371 ± 35 g; excretion rate = 9.0 ± 1.0 ~g of biliru~in per 10 min) was ~ake~ as an. estimate of cycling rate of bilirubin from and back into the pentoneal cav1ty, whiCh was occurnng 1mmedwtely before bile duct translocation .
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CASE REPORTS
100
i.
.e
150
c c c
1 :a
100
j 50
• 3>"
500
1000
2000
( ~g/hr/rol)
FIG. 2. Correlation of serum bilirubin content with rate of absorptive cycling or intravenous infusion of bilirubin. Abscissa, estimated rate of cycling of bilirubin in rats effusing bile into the peritoneal cavity or rate of intravenous infusion of "H-bilirubin conjugates in normal rats;O, individual data derived from rats effusing bile into the peritoneal cavity 19 to 48 hr (summarily shown in table 6) ; o, data derived from normal bile fistula rats infused intravenously with exogenous "H-bilirubin conjugates.
absorption of bilirubin from the peritoneal cavity should be expected, although it has not been previously described. In our normal rats, labeled bilirubin conjugates and unconjugated bilirubin were both rapidly absorbed from the peritoneal space. Thus, the mechanism of absorption across the peritoneum seems to resemble the endothelial membranes (i.e., glomerulus) 27 rather than the epithelial membranes (i.e., intestine, gall bladder, placenta, and renal tubules). 12' 28 In the Gunn rats, bilirubin conjugates passed through the peritoneal membranes and were excreted in the bile as rapidly as in the normal rats. This suggested that most bilirubin glucuronides were not hydrolyzed to free bilirubin when they were absorbed from the peritoneal cavity. Such deconjugation would have resulted in the retention of bilirubin in the serum of the Gunn rat and seriously impaired excretion into the bile!• · 17 In rats effusing bile into the peritoneal
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cavity for 48 hr by way of a polyethylene cannula, only mild liver damage was indicated; BSP clearance was 91 % of normal controls, and aH-bilirubin injected intraperitoneally appeared in the serum and liver at normal rates. However, the possibility that hepatic impairment of normal bilirubin clearance contributed to hyperbilirubinemia was not excluded. Serum volumes were mildly depleted (91 % of normal) and some hemoconcentration occurred (118%); both phenomena, when severe, have been previously shown to result in reduced hepatic perfusion and electron microscopically demonstrable hepatic injury. 29 ' :!0 Bile salts absorbed from the peritoneal space may have accelerated red blood cell destruction and thereby increased hyperbilirubinemia . Obstructive effect of pressure on the common bile duct in the closed, distended ascitic space could have contributed to the hyperbilirubinemia, but this was not indicated; biliary excretion of BSP into the peritoneal cavity was as rapid as it was when biliary flow was exteriorized. Despite the above possibilities, we observed that the intravenous infusion of labeled bilirubin in normal animals at rates identical to estimated peritoneal absorption rates produced hyperbilirubinemia quantitatively identical to that observed in animals effusing bile into the peritoneal cavity. Thus, hyperbilirubinemia in rats effusing bile into the peritoneal cavity appeared to be due to cycling of the extravasated bilirubin from and back into the peritoneal cavity. The effect of bile salts on cycling of bilirubin in these rats is not known. In our patient, the quantitative contribution of peritoneal absorption of bilirubin to hyperbilirubinemia could not be established since experiments comparable to those performed in animals were not possible. It appeared that biliary flow was variably diverted into the peritoneal cavity during the 26 days before surgical procedure. The rapid rise in serum bilirubin on days 13 to 16 may have been prompted by pressure in the distended peritoneal cavity on the bile duct and on the adhesions, tending to wall off the extravasating
November 1970
CASE REPORTS
bile at its source.: 11 As indicated in our animal studies, the rise could have been intensified by absorption of bilirubin from the intraperitoneal pool. The drop in serum bilirubin after paracentesis on day 16 was probably due to decompression in the peritoneal cavity, since no additional trauma to the patient was reported. It was not likely that the removal of bilirubin from the peritoneum was responsible for the drop, particularly in view of the failure of serum bilirubin to increase again during the 8 subsequent days of continued cholascos. REFERENCES 1. Fletcher WS, Mahnke DE, Dunphy JE: Complete division of the common bile duct due to blunt trauma: Report of a case and review of the literature. J Trauma 1:87-95, 1961 2. Dorton HE: Complete division of the common bile duct due to blunt trauma: Case report. Amer Surg 31:333-335, 1964 3. Shelby JS, Cotlar AM, Massari F, et al: Antibiotics in experimental bile peritonitis. Surg Forum 11:305-30fi, 1960 4. Barrett PVD, Berk PD , Menken M, et al: Bilirubin turnover studies in normal and pathologic states using bilirubin-"C. Ann Intern Med 68:355-377, 19()8 5. Carbone JV, G ;·odsky GM: Constitutional nonhemolytic hyperbilirubinemia in the rat: Defect of bilirubin conjugation. Proc Soc Exp Bioi Med 94:461-463, 1957 6. Davidson JD, Feigelson P: Practical aspects of internal-sample liquid scintillation counting. Int J Appl Radiat 2:1-18, 1957 7. Ostrow JD, Hammaker L, Schmid R: The preparation of crystalline bilirubin-C' ' . J Clin Invest 40: 1442- 1452, 19fil 8. Malloy HT, Evelyn KA: Determination of bilirubin with the photoelectric colorimeter . J Bioi Chern 119:481- 490, 1937 9. Grodsky GM, Carbone JV, Fanska R, et al: Tritiated bilirubin: Preparation and physiological studies. Amer J Physiol 203:532-536, 1962 10. Arias IM, Johnson L, Wolfson S: Biliary excretion of injected conjugated and unconjugated bilirubin by normal and Gunn rats. Amer J Physiol 200:1091-1094, 1961 11. Callahan EW Jr, Schmid R: Excretion of unconjugated bilirubin in the bile of Gunn r ats. Gastroenterology 57:134- 137, 1969 12. Ostrow JD: Absorption of bile pigments by the gall bladder. J Clin Invest 46:2035-2052, 1957 13. Schoenfeld LJ, Bollman JL: Further studies on the nature and source of the conjugated bile
777
pigments. Proc Soc Exp Bioi Med 11 2:929-9:12, 1963 14. Weber AP, Schalm L, Whitmans .J: Bilirubin monoglucuronide (pigment 1): A complex. Acta MedScand 173:19- 24, 19(i:l 15. Billing BH, Cole PG, Lathe GH, The exc reti on of bi lirubin as a diglucuronide giving the direct van den Bergh reaction. Biochcm .J G5:774-784, 1957 16. Grodsky G: Studies in the upL1kc and intrahepatic transport of ('H) bilirubin, sect :J, Bi lirubin Metabolism. Edited by lAD Bouchier, RH Billing. Oxford, Blackwell Scientific Publications, 1967, p 159- 169 17. Schmid R, Axelrod ,J, Hammaker L, ct. a l: Congenital jaundice in rats, due to a defect in glucuronide formation. ,J Clin Invest :l7 :11 2:3- li:JO, 1958 18. Bollman JL, Cain ,JC, Grindlay ,JH, Techniques for the collection of lymph from the liver, sma ll intestine, or thoracic duct of the rat. ,J Lab Clin Med 33 :1349-1352, 1948 19. Job TT: The adult anatomy of the lymphatic system in the common rat (Epimys norve{ficus). Anat Rcc 9:447-4!i8, 1915 20. Olin T, Saldeen T, The lymphatic pathways from the peritoneal cavity; A lymphangiograph ic study in the rat. Cancer Res 24:1700- 1711 , l9fi4 21. Gaebler OH: Determination of bromsulphalein in normal, turbid, hcmoly~ed or icteric serums. Amer J Clin Path l5:4!i2- 4/ifJ, 1945 22. Everett NB, Simmons B, Lasher EP: Distribution of blood (Fe''") and plasma (!"") volumes o f rats determined by liquid nitrogen freezing . Circ Res 4:419-424, 19!i6 23. Carbone JV, Grodsky GM, Fanska R : Recent advances in bilirubin metabolism , chap l!i, Current Concepts of C linical Gastroenterology. Edited by .JR Gamble, DL Wilbur. Boston, Little, Brown and Co, Inc, l95(i, p 2o:J- 216 24 . Grodsky GM: Aspects of the metabolism of bromsulfalein and bilirubin, sec :J, Parinatal Pharmacology, Report of the Forty-first Ross Conference on Pediatric Research. Edited by CD May. Columbus, Ohio, Hoss Laboratories, 1962, p 68-78 25. Courtice FC, Simmonds WJ: Physiological significance of lymph drainage of the serous cavities and lungs. Physiol Rev 34:419-448, 1954 26. Boen ST: Kinetics of peritoneal dialysis: A comparison with the artificia l kidney. Medicine (Bait) 40 :243-287, 1961 27 . Schanker LS: Passage of drugs across body membranes. Pharmacal Rev 14:501-530, 1962 28. Lester R, Schmid R: Intestinal absorption of bile pigments I. The enterohepatic circulation of bilirubin in the rat. J Clin Invest 42:736-746, 1963 29. Thoren L : Bile peritonitis: An experimental
778
CASE REPORTS
study on rabbits. Acta Chir Scand (suppl) 230:1106, 1958 30. Albot G, Albot MP, Etienne JP, et al: Signification due syndrome dit "de cholostase" role du !'intoxication biliaire II. Ultrastructure due foie dans !'intoxication biliaire par choleperitoine
Vol. 59, No. 5
total chez le rat, sec 2, Advances in Hepatology. Edited by J Vandenbroucke, J De Groote, LO Standaert. Baltimore, The Williams & Wilkins Co, 1965, p 63-79 31. Sanner RF Jr: Chronic idiopathic bile peritonitis. JAMA 194:1140-1142, 1965
Vol. GH, No. ;,
Copyright © 1970 by The \Villiams & Wilkins Co,
11rintecl in l!. .0...,'. A.
CASE REPORTS
JAUNDICE ASSOCIATED WITH PERITONEAL ABSORPTION OF BILIRUBIN P.
STATIIAKOS,
G.
M.
M.D., R PH.D.
FANSKA, B.S.,
,J. V.
CAIUIONE,
M.D.,
AND
GHODSKY,
Division of Gastroenterology, Department of Medicine, The Permanente Medical Group, Kaiser Foundation Hospital, Oahland, and the Department of Biochemistry and Biophysics, the Department of Medicine, and the Metabolic Research Unit of the Department of Medicine, University of California School of Medicine, San Francisco, California
Jaundice was observed in a patient whose common bile duct was completely severed for 26 days. Absorption of the extravasated bilirubin and varying diversion of biliary flow into the peritoneal cavity contributed to the icterus. Because peritoneal absorption of bilirubin had not been previously defined, studies were conducted in the rat. In bile fistula and lymph fistula rats, absorption was determined by the intraperitoneal injection of aH-bilirubin or by application of the isotopic pigment on peritoneal membranes. Unconjugated bilirubin and bilirubin conjugates were both rapidly absorbed. Studies in the Gunn rat indicated that bilirubin conjugates could pass the peritoneal membranes without being converted to unconjugated bilirubin. Absorption by way of the thoracic lymph accounted for about 11 'X, of the peritoneal absorption; the remainder was presumably absorbed by way of the diaphragmatic lymph and by diffusion through membranes of the parietal and visceral peritoneum. In rats in which the common bile duct was transected, overgrowth of the duct by adhesions stopped biliary flow into the peritoneal cavity within 24 hr. Alternatively, a polyethylene cannula was inserted into the bile duct. With bile freely effusing into the peritoneal cavity for periods of 19 to 48 hr, serum bilirubin was elevated. Most of the hyperbilirubinemia was found to be due to the rapid cyclic absorption of bilirubin from the peritoneal space and back into it sequentially by way of the blood, liver, and bile duct. Jaundice in the patient is discussed in view of the findings in the rat. A number of conditions are known to result in the excretion of bile into the peri-
toneal cavity: idiopathic infantile bile peritonitis, spontaneous idiopathic bile peritonitis, and blunt trauma to the abdo-
Received June 20, 1969. Accepted June 2, 1970. Address requests for reprints to: Dr. G. M. Grodsky, Metabolic Research Unit, University of Califor· Group, Kaiser Foundation Hospital, Oakland, Cali· nia School of Medicine, San Francisco, fornia, for referring the case for further study; H. California 94122. Stathakos for technical assistance; Barbara Haas, This investigation was supported by United Norma Brust, and K. Nemechek, of the University States Public Health Service Grant A-6115 and by of California Medical Center, for technical assist· Kaiser Foundation Hospital Grant 672-0 (66). ance; and Dr. Rudi Schmid, University of California The authors thank Dr. Harris H. Holboe of the Medical Center, for supplying Gunn rats used in this Department of Surgery, The Permanente Medical study and for review of the manuscript. 769
770
CASE REPORTS
men. Complete severance of the common bile duct resulting from blunt trauma to the abdomen, a rare event, is uniformly fatal without surgical intervention. 1- 3 In such a patient, hyperbilirubinemia was observed. There was also high concentration of bilirubin in the ascitic fluid. The absorption and disposition of this bilirubin had not been previously defined. In view of the possibility that the jaundice could have arisen from the absorption of bilirubin from the ascitic fluid, studies were conducted in the rat.
Case Report In a 19-year-old male, the common bile duct was completely severed by blunt trauma incurred in an automobile accident on day 1. By day 10 his abdomen was slightly distended, and serum bilirubin was elevated (fig. 1) . On days 13 to 16, serum bilirubin rapidly rose. On day 16, abdominal paracentesis yielded 8 liters of jaundiced fluid. The following day, serum bilirubin was lowered, presumably a result of the abdominal tap. No additional trauma to the patient was reported on day 16. Serum bilirubin remained essentially unchanged at the ' lower level until day 27, when the completely transected bile duct was transplanted to the duodenum; adhesions were seen between the liver and the abdominal wall. Mter the surgical procedure, serum bilirubin became normal and the patient returned to good health. During the 26 days before operation, alkaline phosphatase was 29 to 44 King-Armstrong
Vol. 59, No . 5
units (days 15 to 24) ; serum glutamic oxaloacetic transaminase was 100 units (day 19) ; Bromsulphalein (BSP) retention was 16% (day 24), and about 7 g of bilirubin were excreted into the peritoneal cavity.•
Materials and Methods Labeled bilirubin in donor bile was injected intraperitoneally or applied on selected sites of visceral and parietal peritoneum in bile fistula and lymph fistula male Long-Evans rats and homozygous (jj) , jaundiced Gunn rats.5 Absorption of labeled pigment was determined by the subsequent excretion of radioactivity in bile and lymph or by assessment of isotope content of serum and liver. In rats with surgically transected bile duct, the relationship of jaundice to the peritoneal absorption of bilirubin was investigated. Counting and crystallizing tritiated bilirubin. Bilirubin isotope was counted in a Packard Tri-Carb liquid scintillation photospectrometer . Lymph (60 "liters), serum (20 "liters) , bile (20 "liters), or homogenized liver (25 mg) was diluted with 0.25 ml of water and freezedried. The dry sample was dissolved in 1.5 ml of a molar solution of Hyamine in methanol in the presence of 90 "liters of 30% hydrogen peroxide and was diluted with 15 ml of scintillator solution. 6 All samples were corrected for quenching by measuring the recovery of "Hglycine added to a duplicate sample. Aliquots of bile containing "H-bilirubin were assayed for degraded isotope by the bilirubin crystallization method of Ostrow eta!. 7 Chemical determination of bilirubin in ser-
e 0
0
0 Days after automobile accident FIG .
1. Serum bilirubin levels in the patient.
Novemb er 1970
C ASE REPORTS
um, bile, and peritoneal fluids. Direct and total bilirubin was determined by the diazo method of Malloy and Evelyn," except that the sulfanilic acid concentration of the reagent was increased 2-fold. Diazo standards were prepared with certified reagent bilirubin (Fisher Scientific Company). Serum was diluted 1:20 with water before reagent was added . Bile and peritoneal fluid were diluted with water so that the amount of bilirubin that reacted with 1 ml of diazo reagent was between 3 and 25 11 g. The maximum optical density for the determination of total bilirubin was obtained from photometric readings made at 3-min intervals. Under these conditions, photometric absorption was linear with bilirubin concentration. Collection of bile. Bile was collected in Wintrobe sedimentation tubes which had been covered with aluminum foil to exclude light and placed in an ice bath; subsequently, bile was frozen until just before use. Preparation of donor bile containing bilirubin conjugates . For injections or infusions in the rats, conjugated bilirubin was obtained from bile fistula donor rats given intravenous injections of 0 _1 M Na2C02 solution of tritiated bilirubin 9 or bilirubin (Eastman) . Injection of 150 11g of "H-bilirubin (specific activity 23 11 c per mg) , repeated hourly, resulted in biliary excretion of approximately 150 Jlg of total bilirubin per ml of bile (specific activity 15 11 c per ml) . A mean of 95% of the radioactivity in the bile was crystallizable "H-bilirubin. Injections of 1 mg of Eastman bilirubin per 100 g body weight at 20-min intervals gave rise to excretion of 8,000 t o 10,000 11g of unlabeled bilirubin per ml of bile . Labeled and unlabeled bile were usually combined and diluted with 0.9'/(, saline. Injectates and infusates contained approximately 1 ml of bile and 1 ml of saline . For paintings on small areas of parietal peritoneum, labeled bile of high specific activity was prepared by injecting 900 11g of "H-bilirubin hourly. This technique has been used for the preparation of labeled bilirubin conjugates in other studies, 10 12 even though the nature of the conjugates has not been established. Normally, most of the bilirubin excreted in bile is bilirubin diglucuronide. 1" However, significant excretion of polar complexes of unconjugated bilirubin with the diglucuronide, 14 or possibly as bilirubin monoglucuronide, can also occur. 15 In ancillary studies, we investigated these biliary products after intravenous injection of 1 mg of labeled bilirubin per 100 g of body weight into normal rats. By the modified diazo technique above, direct reacting bilirubin was 99 to 100% of the total. Paper chromatography
771
of the undiazotized pigments in n-propanol, nbutanol, and wa ter (3: 4: 3) at 4 C and in nitrogen atmosphere 1 ,; gave two major bands. More than 50'/;, of the total pigment was in a band correspondin g in mobility and glucuronide content to bilirubin diglucuronide. Most of the remainder was distributed into a less polar band containing variably less than 2 moles of glucuronic acid per mole of bilirubin, but with mobility different from lipophilic unconju gated bilirubin, the latter comprising less than 4% of the total pigment. In the present study, these normal biliary excretion products of bilirubin are referred to as bilirubin conjugates. Their absorption was often tested in the Gunn rat to establish whether absorbed tritiated pigment was or was not unconjugated bilirubin. Absorbed, unconjugated "H-bilirubin in the circulation of the Gunn rat would not be secreted by the liver and appear in the bile in amounts comparable to absorbed conjugated forms." Preparation of unconjugated bilirubin solutions for intraperitoneal injection in the rat. This was mad e by dissolving bilirubin and "Hbilirubin in 0.1 M Na2CO" to final concentration of 2 mg per mi. The pH was adjusted to 9.0 with 0.5 M HCl, and an equal volume of normal rat bile was added. Further dilution when required was made with 0.9'/(, saline . Biliary excretion of intraperitoneally injected "H-bilirubin conjugates and unconjugated bilirubin. After 1-cm midline incision in normal and Gunn rats, PE 50 polyethylene tubing (Clay Adams, Division of Beckton, Dickinson and Company) was introduced into the common bile duct. With the distal end of the cannula exterior to the rat, peritoneum, muscle, and skin were each sutured. After injection of donor bile containing labeled bilirubin conjugates or unconjugated bilirubin, recipient bile was collected at 1-hr intervals for 2 hr. Each bile was assayed for crystallizable isotope content. Excretion of intraperitoneally injected "Hbilirubin conjugates in thoracic lymph and bile. Normal rats received high fat diet for 24 hr before the thoracic duct was cannulated as previously des cribed by Bollman et al. 1" Each animal was placed in a Bollman restraining cage where it was fed stock rat diet and watered with 0.9 % NaCl ad libitum. The following day, each rat was removed from the cage and the common bile duct was cannulated. After injection of donor bile containing labeled bilirubin conjugates, recipient lymph and bile were collected at hourly intervals for 2 hr. Tritium in lymph and bile was counted, and "H-
772
CASE REPORTS
bilirubin in bile was crystallized. Since cannulation of the thoracic duct was above the cisterna chyli, virtually all lymphatic drainage from the peritoneal cavity, except that by way of the diaphragmatic lymph, was collected. 19 ' 20 Absorption of 3 H-bilirubin conjugates by the visceral and parietal peritoneum . Absorption by the visceral peritoneum was assessed in normal rats. The common bile duct was cannulated. A midline incision was lengthened to allow extrusion of the intestine. The rat was positioned prone over a small beaker containing 2000 11 g of bilirubin conjugates (labeled with 2 11c of tritium) in 1 ml of bile diluted with 4 ml of saline. The ileum, cecum, and 1 em of colon were bathed. Bile was collected for 2 hr in the dark. Absorption by the abdominal wall (parietal peritoneum, drained by thoracic lymph) was demonstrated in normal and in Gunn rats. The bile duct was cannulated and the midline incision was extended from the xiphoid process to the pubis. From the ends of the incision, diagonal cuts were made which produced bilateral flaps of abdominal wall. The flaps were pulled to the sides of the rat by means of hemostats. A clamp composed of several thicknesses of aluminum foil was used to elevate the viscera above the flaps . In a darkened room, bile containing bilirubin conjugates was rolled onto the flaps with a cotton-tipped applicator at 7min intervals, over a period of 2 hr. Bile was collected during this period. The surface area of the parietal peritoneum was measured from imprints of the surfaces which were made on graph paper. Absorption by the diaphragm (parietal peritoneum, drained by diaphragmatic lymph) was shown in normal rats. The bile duct was cannulated and abdominal flaps were constructed as described above. Bile containing bilirubin conjugates was rolled onto the ventral diaphragm at intervals of 7 min, with the rat inclined 15°, head down. The quantity of bilirubin applied on the diaphragm or abdominal wall was determined from the difference between bilirubin originally assayed in the solution from which application was made and the sum of bilirubin (a) remaining in the solution, (b) on the applicator, and (c) rinsed from the topical site. The bile of all rats was assayed for isotope content and "H-bilirubin was crystallized from it. Relationship of jaundice to the peritoneal absorption of bilirubin in rats effusing bile into the peritoneal cavity. In 6 normal rats, the abdomen was opened, the common duct was
Vol. 59, No . 5
transected at 1 em from its proximal end, and the peritoneum, muscle, and skin were each sutured. Mter 24 hr, exploratory laparotomy was performed to ascertain the reasons for the failure of fluid to accumulate in the peritoneal cavity, and the rats were killed. In the remainder of the rats, biliary effusion was prolonged by cannulating the bile duct with PE 50 polyethylene tubing. The cannula was clipped to 1cm length, and the distal tip was placed in the peritoneal cavity. The incision was sutured as above. The a nimals were allowed routine diet and water ad libitum without supportive therapy for periods up to 48 hr. Liver function and bile duct patency were assessed in 6 rats in which effusion was prolonged. Bromsulphalein, 5 mg per kg preoperative body weight, was injected intravenously while the cannula remained in the peritoneal cavity, or after midline incision was reopened to permit extension of the cannula to the exterior of the rat by forcing it into tightly fitting PE 90 tubing. The dye was assayed in bile or peritoneal f1uid by the method of Gaebler" 45 min after injection (in control rats, the common bile duct was cannulated; bile flow was directed either intraperitoneally or outside of the body, and BSP was injected without delay). Serum volume was determined from dilution of intravenously injected "11 1 human serum albumin. 22 Peritoneal absorption was assessed in 6 rats effusing bile into the peritoneal cavity by the appearance of label in serum and liver, after bile containing 3 H-bilirubin conjugates was injected intraperitoneally. Ten minutes after injection, the rats were decapitated and tritium content of serum and liver was determined. During this short period, most absorbed pigment can be accounted for in the serum and liver, since secretion into the bile does not have time to occur 23 (in an ancillary study, no surgery was performed before "H-bilirubin conjugates were injected). Cycling of endogenous bilirubin from and back into the peritoneal cavity was estimated in 9 rats effusing bile into the peritoneal cavity. The midline incision was reopened, and the cannula was extended outside the body. Bile was collected for 10 min, and each rat was decapitated. The rate of excretion of endogenous bilirubin in the bile and the bilirubin content of serum and peritoneal fluid were determined by the diazo reaction. Intrav enous infusion of "H-bilirubin conjugates in bile fistula rats to achieve hyperbilirubinemia comparable to that in rats effusing bile into the peritoneal cavity. Bile containing
November 1970
CASE REPORTS
labeled bilirubin conjugates in 6 different concentrations was infused into the inferior vena cava for 90 min . Serum and bile were assayed for tritium content at 15-min intervals during infusion. The infusion technique has been pre24 viously described.
Results Biliary excretion of intraperitoneally injected 3 H-bilirubin conjugates and unconjugated :1H-bilirubin. Rapid excretion of crystallizable isotope is shown in table 1. After injection of either 3 H-bilirubin conjugates or :1H-unconjugated bilirubin in normal rats, about 20% of the dose was recovered in bile by 2 hr; the rate of excretion was slightly higher during the second hour. 3 H-bilirubin conjugates were excreted in the bile of Gunn rats and normal rats at an equal rate. A mean of 94% of the isotope in the bile of the rats given injections of bilirubin conjugates was crystallizable. This indicated molecular integrity of excreted aH-bilirubin. When unconjugated "H-bilirubin was injected, a
mean of 89 % of the isotope appearing in bile was crystallizable. The mean bile volume excreted by the normal rats of the group was 0.93 ml per hr; the Gunn rats excreted 0.57 ml per hr. Excretion of intraperitoneally injected aH-bilirubin conjugates in thoracic lymph and bile. Rate of excretion in lymph and bile is shown in table 2. During the first 2 hr, label excreted in lymph and bile was 91 % of that excreted by rats with bile fistula only (table 1). Ninety-three per cent of the isotope was crystallizable from bile in this experiment. Approximately 11 (X, of the radioactivity excreted was through the lymphatic system. In contrast to bile, this pathway became less important during the second hour. After intraperitoneal injection, mean volumes of lymph and bile were 1.5 and 0.7 ml per hr, respectively. Absorption of aH-bilirubin conjugates by the visceral and parietal peritoneum. Rapid absorption by the visceral peritoneum when bathed in a solution of bilirubin conjugates was indicated as follows:
Biliary excretion of intraperitoneally injected "H-bilirubin conJulfates and unconjulfaled "Hbilirubin ( CB and UCB) in the rat"
1.
TABLE
773
1
No. of
Body
rats
weight
:
1
H -Biliruhin injected into peritoneal cnvity
i-I-Biliruhin excreted in hilc
Dur ing the lin;t hour nflcr injection
~ ·;. ofdww
I!
Normal 3 3 Gunn 3
397 ± 5 333 ± 17
CB (in bile) UCB (added to bile)
7.7 ± 0.6 8.3 ± 0.5
9.8 ± 0.4 10.6 ± 0.3
207 ± 2
CB
7.4 ± 0.5
9.7 ± 0.3
' Mean values (±sE) are shown. Quantity of bilirubin injected in all rats was 2000 of tritium. Volume injected was 2.0 mi. TABLE
2.
1 :
rats
Body weight
g
3
~g.
labeled with 2.0
~c
Excretion of intraperitoneally injected "H-bilirubin conjugates in thoracic lymph and bile of the rat' Qunntityof
No . of
During the second hour after injcclion
342 ± 14
1 :
H-hilirubin
injected into peritoneal cav ity
2~0
I
In bile
2.0
I
During the
During the first hour after injection
During the second hou r nftcr injection
nftcr injection
1.1 ± 0.5
0.6 ± 0.2
6.1 ± 0.8
first hour ~·;.
ml
pC
1 2.0
H-Bilimbin excreted
In lymph
During the second hour after injection
of dose
'f
8.2 ± 0.9
'Mean values (±sE) are shown. A cannula was placed in the abdominal thoracic duct and another in the common bile duct of each rat before injection.
774
CASE REPORTS
mean biliary excretion of crystallizable H-bilirubin absorbed by the ileum and cecum was 1.5% of the dose in the first hour and 1.6% in the second hour. These represent minimal values, since the surfaces bathed were visually estimated to be one-half those of the entire visceral peritoneum. A mean of 94% of the radioactivity excreted in bile was crystallizable 3 H-bilirubin. Rapid absorption of 3H-bilirubin conjugates by the parietal peritoneum in normal and Gunn rats is shown in table 3. In the Gunn rats, bilirubin conjugates were also rapidly absorbed by the abdominal wall and excreted in the bile. A mean of 87% of the radioactivity excreted in bile was crystallizable 3 H-bilirubin. The relationship of jaundice to the peritoneal absorption of bilirubin in rats effusing bile into the peritoneal cavity. In all of the rats in which a cannula was not placed in the bile duct, biliary flow into the peritoneal cavity ceased within 24 hr. Complete overgrowth of the duct by adhesions was seen. Results in the remaining
rats of this group were obtained by inserting a cannula into the severed bile duct. In a series of rats with 48-hr of effusion of bile into the peritoneal cavity, the percentage of intravenous dose of BSP found in ascitic fluid after 45 min was 68 ± 3 (n = 6); when bile flow was exteriorized, 84 ± 1% (n = 6) of the dose appeared in the bile. In either case, the mean value was 91 % of that found in control rats. In all rats having exteriorized bile cannula, BSP was not detected in ascitic fluid . These results indicated patency of the bile duct and minimal liver damage in rats with 48 hr of effusion of bile into the peritoneal cavity. Serum volume was 3.04 ± 0.08 ml per 100 g of preoperative body weight (n = 10), approximately 91 % of that in normals; hematocrit was 118% of normal. Large amounts of endogenous bilirubin were found in the peritoneal cavity by 48 hr (table 4). The animals had mild hyperbilirubinemia of 0.9 mg per 100 ml. Mean bilirubin concentration in peritoneal fluid was 12.8 times that in serum. The rate of absorption of a trace intraperitoneal dose
3
TABLE
No. of rats
Biliary excretion of 'H-bilirub in conjugates topically painted on the parietal peritoneum"
3.
aH·Bilirubin topically pain ted on parietal peritoneum
Body weight
During the Quantity
400 ± 17 390 ± 5
Gunn 3
207 ± 7
Abdominal wall Ventral diaphragm Abdominal wall
During the seco nd hour after injection
first hour
after injection
em '
li
6
:'H-Bi liruhin excreted in bile
Locus Total a rea painted
Normal 6
Vol . 59, No.5
~;, of
"c
1'1!
do.'ie
14
3050 ± 182
3. 0 ± 0. 2
4.7 ± 1.4
8.3 ± 0.5
4
3200 ± 278
3.2 ± 0.3
9. 1 ± 1.1
9.6 ± 1.3
10
2100 ± 200
2.1 ± 0.2
4.6 ± 1.5
8 .2 ± 1.4
" Mean valu es (±SE) are shown . The mea n surface area of the abdominal wall was 8.2 times the total area painted.
TABLE
4.
Distribution of endogenous bilirubin in rats in which the common bile duct was severed for 48 hr" Body weight
No. of rats
N ormal
l
Peritoneal fluid
370 ± 25
j
Volume
Total bilirubin
48 hr
Volume ml
,g
ml
,g
380 ± 27
25± 7
2980 ± 644
11.3 ± 0.3
105 ± 13
g
10
Serum
T ota l bilirubin
" Mean values (± sE) are shown. Normal serum contai ns less than 10 11g of total bilirubin.
November 1970
CASE REPORTS
775
3
of H-bilirubin conjugates was about equal to or greater than that in normal unoperated rats (table 5). Rates of cycling of endogenous bilirubin after 19 to 48 hr of bile effusion into the peritoneal cavity are shown in table 6. Excretion rate of bilirubin during the first 10 min after exteriorizing the bile duct minus normal bilirubin output in bile (normal bile fistula rats, n = 9, body weight = 371 ± 35 g; excretion = 9.0 ± 1.0 11 g bilirubin per 10 min) was taken as an estimate of cycling rate of bilirubin from the peritoneal space, through blood, liver, and bile, which was occurring immediately before bile duct translocation. Excretion rate during this time was about 15 times normal. Intravenous infusion of :1H-bi lirubin conjugates in bile fistula rats to achieve hyperbilirubinemia comparable to that in rats effusing bile into the peritoneal cavity. Regardless of the dose infused,. comparatively steady concentrations of 3 H-biTABLE
5.
lirubin serum (and bile) were reached before 90 min. In each of 6 rats, serum bilirubin content at 90 min was plotted on a graph against the infusion rate, together with serum bilirubin content and estimated cycling rate of each of 9 rats (summarily shown in table 6) effusing bile into the peritoneal cavity 19 to 48 hr. The result is shown in figure 2. Although the state of the rats in these two groups was quite different, a closely parallel linear relationship was obtained on the graph. Thus, most hyperbilirubinemia in rats in which the bile duct was severed appeared to be due to absorptive cycling of endogenous bilirubin. Discussion Comprehensive reviewsir.. il; indicate that many molecules rapidly pass through the peritoneal membranes in man and animals without distinction as to lipid solubility and molecular charge. Thus,
Rates of absorption of intraperitoneally injected "H-bilirubin conju~:ates in normal rats and rats effu~ing bile into the peritoneal cavity for 48 hr~" Normal body
No. of rats
weight
Qunntity of :t H-hiliruhin conjugates injected into pcritoncnl cnvity
1
Appenl"nnce of ' H-hilirubin
In serum
Serum
+
liver
I'll
pC
ml
369 ± 12
150
2.0
2.0
1.5 ± 0.1
2.5 ± 0.2
4.0 ± 0.3
365 ± 24
150
2.0
2.0
2.3 ± 0. 3
2.9 ± 0.4
5.2 ± 0.7
~
Normal, without surgery 6 Effusing bile for 48 hr 6
In liver
Hate of nhsorpti
.'j, of dose/10 min
1
' Mean values (±sE) are shown. TABLE
6.
Rate of cycling of endogenous bilirubin in rats effusing bile into the peritoneal cavity for 19 to 48 hr" Distribution of bilirubin after bile flow direc ted cxtraperitoncally during last 10 min of final hour
No. of rats
9
Normal body weight
In peritoneal fluid
II
I'll
370 ± 35
2320 ± 1182
Excreted in
In serum
Cyclinf.! of bilirubin
bile
~
w:/10 min
76 ± 31
146 ± 34
..
~«110
min/rat
137 ± 32
..
' Mean values (±sE) are shown . Bile duct transected 33 ± 10 hr. Bil1ary excretwn rate of b1hrubm while the bile duct was external to the rat minus bilirubin output in normal bile fistula rats (n = . 9, body weight = 371 ± 35 g; excretion rate = 9.0 ± 1.0 ~g of biliru~in per 10 min) was ~ake~ as an. estimate of cycling rate of bilirubin from and back into the pentoneal cav1ty, whiCh was occurnng 1mmedwtely before bile duct translocation .
776
CASE REPORTS
100
i.
.e
150
c c c
1 :a
100
j 50
• 3>"
500
1000
2000
( ~g/hr/rol)
FIG. 2. Correlation of serum bilirubin content with rate of absorptive cycling or intravenous infusion of bilirubin. Abscissa, estimated rate of cycling of bilirubin in rats effusing bile into the peritoneal cavity or rate of intravenous infusion of "H-bilirubin conjugates in normal rats;O, individual data derived from rats effusing bile into the peritoneal cavity 19 to 48 hr (summarily shown in table 6) ; o, data derived from normal bile fistula rats infused intravenously with exogenous "H-bilirubin conjugates.
absorption of bilirubin from the peritoneal cavity should be expected, although it has not been previously described. In our normal rats, labeled bilirubin conjugates and unconjugated bilirubin were both rapidly absorbed from the peritoneal space. Thus, the mechanism of absorption across the peritoneum seems to resemble the endothelial membranes (i.e., glomerulus) 27 rather than the epithelial membranes (i.e., intestine, gall bladder, placenta, and renal tubules). 12' 28 In the Gunn rats, bilirubin conjugates passed through the peritoneal membranes and were excreted in the bile as rapidly as in the normal rats. This suggested that most bilirubin glucuronides were not hydrolyzed to free bilirubin when they were absorbed from the peritoneal cavity. Such deconjugation would have resulted in the retention of bilirubin in the serum of the Gunn rat and seriously impaired excretion into the bile!• · 17 In rats effusing bile into the peritoneal
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cavity for 48 hr by way of a polyethylene cannula, only mild liver damage was indicated; BSP clearance was 91 % of normal controls, and aH-bilirubin injected intraperitoneally appeared in the serum and liver at normal rates. However, the possibility that hepatic impairment of normal bilirubin clearance contributed to hyperbilirubinemia was not excluded. Serum volumes were mildly depleted (91 % of normal) and some hemoconcentration occurred (118%); both phenomena, when severe, have been previously shown to result in reduced hepatic perfusion and electron microscopically demonstrable hepatic injury. 29 ' :!0 Bile salts absorbed from the peritoneal space may have accelerated red blood cell destruction and thereby increased hyperbilirubinemia . Obstructive effect of pressure on the common bile duct in the closed, distended ascitic space could have contributed to the hyperbilirubinemia, but this was not indicated; biliary excretion of BSP into the peritoneal cavity was as rapid as it was when biliary flow was exteriorized. Despite the above possibilities, we observed that the intravenous infusion of labeled bilirubin in normal animals at rates identical to estimated peritoneal absorption rates produced hyperbilirubinemia quantitatively identical to that observed in animals effusing bile into the peritoneal cavity. Thus, hyperbilirubinemia in rats effusing bile into the peritoneal cavity appeared to be due to cycling of the extravasated bilirubin from and back into the peritoneal cavity. The effect of bile salts on cycling of bilirubin in these rats is not known. In our patient, the quantitative contribution of peritoneal absorption of bilirubin to hyperbilirubinemia could not be established since experiments comparable to those performed in animals were not possible. It appeared that biliary flow was variably diverted into the peritoneal cavity during the 26 days before surgical procedure. The rapid rise in serum bilirubin on days 13 to 16 may have been prompted by pressure in the distended peritoneal cavity on the bile duct and on the adhesions, tending to wall off the extravasating
November 1970
CASE REPORTS
bile at its source.: 11 As indicated in our animal studies, the rise could have been intensified by absorption of bilirubin from the intraperitoneal pool. The drop in serum bilirubin after paracentesis on day 16 was probably due to decompression in the peritoneal cavity, since no additional trauma to the patient was reported. It was not likely that the removal of bilirubin from the peritoneum was responsible for the drop, particularly in view of the failure of serum bilirubin to increase again during the 8 subsequent days of continued cholascos. REFERENCES 1. Fletcher WS, Mahnke DE, Dunphy JE: Complete division of the common bile duct due to blunt trauma: Report of a case and review of the literature. J Trauma 1:87-95, 1961 2. Dorton HE: Complete division of the common bile duct due to blunt trauma: Case report. Amer Surg 31:333-335, 1964 3. Shelby JS, Cotlar AM, Massari F, et al: Antibiotics in experimental bile peritonitis. Surg Forum 11:305-30fi, 1960 4. Barrett PVD, Berk PD , Menken M, et al: Bilirubin turnover studies in normal and pathologic states using bilirubin-"C. Ann Intern Med 68:355-377, 19()8 5. Carbone JV, G ;·odsky GM: Constitutional nonhemolytic hyperbilirubinemia in the rat: Defect of bilirubin conjugation. Proc Soc Exp Bioi Med 94:461-463, 1957 6. Davidson JD, Feigelson P: Practical aspects of internal-sample liquid scintillation counting. Int J Appl Radiat 2:1-18, 1957 7. Ostrow JD, Hammaker L, Schmid R: The preparation of crystalline bilirubin-C' ' . J Clin Invest 40: 1442- 1452, 19fil 8. Malloy HT, Evelyn KA: Determination of bilirubin with the photoelectric colorimeter . J Bioi Chern 119:481- 490, 1937 9. Grodsky GM, Carbone JV, Fanska R, et al: Tritiated bilirubin: Preparation and physiological studies. Amer J Physiol 203:532-536, 1962 10. Arias IM, Johnson L, Wolfson S: Biliary excretion of injected conjugated and unconjugated bilirubin by normal and Gunn rats. Amer J Physiol 200:1091-1094, 1961 11. Callahan EW Jr, Schmid R: Excretion of unconjugated bilirubin in the bile of Gunn r ats. Gastroenterology 57:134- 137, 1969 12. Ostrow JD: Absorption of bile pigments by the gall bladder. J Clin Invest 46:2035-2052, 1957 13. Schoenfeld LJ, Bollman JL: Further studies on the nature and source of the conjugated bile
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pigments. Proc Soc Exp Bioi Med 11 2:929-9:12, 1963 14. Weber AP, Schalm L, Whitmans .J: Bilirubin monoglucuronide (pigment 1): A complex. Acta MedScand 173:19- 24, 19(i:l 15. Billing BH, Cole PG, Lathe GH, The exc reti on of bi lirubin as a diglucuronide giving the direct van den Bergh reaction. Biochcm .J G5:774-784, 1957 16. Grodsky G: Studies in the upL1kc and intrahepatic transport of ('H) bilirubin, sect :J, Bi lirubin Metabolism. Edited by lAD Bouchier, RH Billing. Oxford, Blackwell Scientific Publications, 1967, p 159- 169 17. Schmid R, Axelrod ,J, Hammaker L, ct. a l: Congenital jaundice in rats, due to a defect in glucuronide formation. ,J Clin Invest :l7 :11 2:3- li:JO, 1958 18. Bollman JL, Cain ,JC, Grindlay ,JH, Techniques for the collection of lymph from the liver, sma ll intestine, or thoracic duct of the rat. ,J Lab Clin Med 33 :1349-1352, 1948 19. Job TT: The adult anatomy of the lymphatic system in the common rat (Epimys norve{ficus). Anat Rcc 9:447-4!i8, 1915 20. Olin T, Saldeen T, The lymphatic pathways from the peritoneal cavity; A lymphangiograph ic study in the rat. Cancer Res 24:1700- 1711 , l9fi4 21. Gaebler OH: Determination of bromsulphalein in normal, turbid, hcmoly~ed or icteric serums. Amer J Clin Path l5:4!i2- 4/ifJ, 1945 22. Everett NB, Simmons B, Lasher EP: Distribution of blood (Fe''") and plasma (!"") volumes o f rats determined by liquid nitrogen freezing . Circ Res 4:419-424, 19!i6 23. Carbone JV, Grodsky GM, Fanska R : Recent advances in bilirubin metabolism , chap l!i, Current Concepts of C linical Gastroenterology. Edited by .JR Gamble, DL Wilbur. Boston, Little, Brown and Co, Inc, l95(i, p 2o:J- 216 24 . Grodsky GM: Aspects of the metabolism of bromsulfalein and bilirubin, sec :J, Parinatal Pharmacology, Report of the Forty-first Ross Conference on Pediatric Research. Edited by CD May. Columbus, Ohio, Hoss Laboratories, 1962, p 68-78 25. Courtice FC, Simmonds WJ: Physiological significance of lymph drainage of the serous cavities and lungs. Physiol Rev 34:419-448, 1954 26. Boen ST: Kinetics of peritoneal dialysis: A comparison with the artificia l kidney. Medicine (Bait) 40 :243-287, 1961 27 . Schanker LS: Passage of drugs across body membranes. Pharmacal Rev 14:501-530, 1962 28. Lester R, Schmid R: Intestinal absorption of bile pigments I. The enterohepatic circulation of bilirubin in the rat. J Clin Invest 42:736-746, 1963 29. Thoren L : Bile peritonitis: An experimental
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study on rabbits. Acta Chir Scand (suppl) 230:1106, 1958 30. Albot G, Albot MP, Etienne JP, et al: Signification due syndrome dit "de cholostase" role du !'intoxication biliaire II. Ultrastructure due foie dans !'intoxication biliaire par choleperitoine
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total chez le rat, sec 2, Advances in Hepatology. Edited by J Vandenbroucke, J De Groote, LO Standaert. Baltimore, The Williams & Wilkins Co, 1965, p 63-79 31. Sanner RF Jr: Chronic idiopathic bile peritonitis. JAMA 194:1140-1142, 1965