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Origin of Fat in Chylous Ascites of Patients with Liver Cirrhosis
Vol. 67, No.5
GASTROENTEROLOGY" 67:878-886, 1974
Copyright © 1974 by The Williams & Wilkins Co.
Printed in U .S .A.
ORIGIN OF FAT IN CHYLOUS ASCITES OF PATIENTS WITH LIVER CIRRHOSIS JUAN
R.
MALAGELADA, FRANK
L.
lBER, AND WILLEM
G.
LINSCHEER
Lemuel Shattuck Hospital, Department of Medicine, Tufts University School of Medicine , Boston, Massachus etts
To investigate the origin of fat in chylous ascites of patients with liver cirrhosis, labeled medium and long chain fatty acids ( [14 C ]octanoic and [3H]oleic acid, respectively) were fed to 3 cirrhotic patients with chylous ascites as well as to two control groups: patients with cirrhosis and clear ascites and noncirrhotic subjects with chylous ascites. The time of appearance, time interval from ingestion to peak levels, and half-life time of decay of labeled long chain fatty acids in ascitic chylomicrons were similar in all groups of patients. In the cirrhotic patients, the peak activity of long chain fat found in chylous ascites (8% of ingested radioactivity) was much greater than in clear ascites (0.6%). An even greater amount of ingested long chain fat was found in the chylous ascites of noncirrhotic patients (22%). More than 80% of the ingested [3 H ]oleic acid present in chylous ascites was incorporated in esterified form in chylomicrons as compared to less than 50% in clear ascitic fluid. This significantly higher (P < 0.01) incorporation of 3H radioactivity in subjects with chylous ascites when serum levels were identical suggests that intestinal lymph was the main source of ascitic chylomicrons. The appearance, concentration, and decay rate in ascites of [14 C ]octanoic acid, not incorporated in the chylomicrons, was similar in cirrhotic patients with clear or chylous ascitic fluid, suggesting a similar pathway of transport of the nonprotein-bound [14 C )octanoic acid. Analysis of the relative concentrations of triglycerides, phospholipids, and cholesterol in ascitic fluid revealed a relatively low proportion of triglycerides in clear ascitic fluid which indicates that most of the triglycerides are present in the smaller particles, probably a combination of small chylomicrons, very low, low, and high density lipoproteins. It can be concluded from our studies that only small chylomicrons, probably leaking from the intestinal lymphatics, enter the ascites of cirrhotic patients with clear ascitic fluid. When the number and size of these chylomicrons increases, the ascitic fluid acquires a chylous nature. Whether the underlying pathogenesis involves abnormal permeability or rupture of the lymphatics is unknown, but the process may reverse with improvement of liver function. Chylous ascites is an infrequent feature of decompensated cirrhosis of the liver. 2 - 5
The origin of fat in ascitic fluid of these patients is unclear. 6 Ascites in cirrhosis is
Received March 2, 1973. Accepted April17, 1974. This work has been reported in part. 1 Address requests for reprints to: Dr. Willem G.
Linscheer, Department of Gastroenterology, Veterans Administration Hospital, Irving Avenue and University Place, Syracuse, New York 13210.
878
November 1974
879
ORIGIN OF FAT IN CHYLOUS ASCITES
thought to be produced by a combination of filtration of fluid through the splanchnic capillary system as a result of portal hypertension and decreases osmotic pressure (low albumin), leakage of lymph from the liver surface (weeping liver), and possibly also from the intestinal lymphatic system. 7 The milky appearance of chylous ascites is thought to be due to the high concentration of chylomicrons. 8 As hepatic lymph contains only a low concentration of chylomicrons, 9 and splanchnic venous capillaries are considered to be impermeable for chylomicrons, 10 the most likely source of chylomicrons in ascites is the intestinal lymph. Although rupture of peritoneal lymph vessel has been demonstrated in patients with lymphatic obstruction due to malignancy, it has seldom been reported in patients with cirrhosis and chylous ascites. This study was designed to investigate the origin of lipids in chylous ascites of cirrhotic patients. As triglycerides are the main constituents of chylomicrons, we have studied the appearance and turnover of ingested 3 H-labeled long chain triglycerides in the ascitic fluid chylomicron fraction. 14 C-labeled medium chain triglycerides, given simultaneously, were used to study transfer of water-soluble soaps from portal and peripheral blood into ascites. Patients with chylous ascites associated with cirrhosis were compared with a group of patients with clear ascites associated with cirrhosis and with a third group of patients with chylous ascites due to lymphatic obstruction. This work was supported in part by Research Grant AM 11566 and Training Grant AM 05424 from the National Institutes of Health, U.S. Public Health Service, and a grant from The John A. Hartford Foundation. Dr. Juan R. Malagelada was a recipient of a Juan March Foundation Fellowship. His present address is: Department of Internal Medicine and Gastroenterology, Mayo Clinic, Rochester, Minnesota 55901. The authors are indebted to Drs. Hyman J . Zimmerman, Leo Stolbach, and Marvin Krant for permission to study patients under their care; to Miss Pat Burns and Miss Sheila Lieberman for technical assistance; and to Dr. Neville Hoffman for his critical review of the manuscript.
Experimental Procedure Patients Fifteen patients divided into three groups were studied (tables 1 and 2). Group 1: cirrhotic patients with chylous ascites (3 patients). All 3 patients were chronic alcoholics in whom the diagnosis of cirrhosis of the liver was established by biopsy. Patient 1, female , 47 years old . One year prior to the study she developed symptoms of cirrhosis with ascites. Her symptoms did not respond to treatment, including diuretics, and she underwent periodic paracentesis to relieve intraabdominal pressure. The ascitic fluid was clear until 10 weeks prior to study when it became progressively milky. No signs of malignancy or mechanical obstruction of the lymphatic system were found at autopsy 2 months after the study. Patient 2, male, 70 years old. Clinical symptoms of cirrhosis appeared 5 years before study; ascites had been present for 18 months . Paracentesis 3 months prior to study revealed chylous ascitic fluid and an abdominallymphangiogram performed at this time showed no abnormality. He died of myocardial infarction 3 months after study. Permission for autopsy was not obtained. Patient 3, male, 42 years old. \1anifestations of cirrhosis with ascites developed at least 8 months before study, but chylous ascites was noted only for the last month. After the study he responded favorably to abstinence froni alcohol, bed rest, and a nutritious diet, and his ascites disappeared completely. He has been followed closely for 1 year and no recurrence of the ascites has been observed. Clinical and laboratory findings at present are consistent with compensated cirrhosis. Group 2: cirrhotic patients with clear ascites (8 patients). The 8 patients, averaging 53 years of age, were all chronic alcoholics in whom cirrhosis was confirmed by liver biopsy, and all had had ascites for a minimum period of 6 weeks. Group 3: noncirrhotic patients with intraabdominal malignancies and chylous ascites (4 patients: two females and two males). Of the 4 patients, who averaged 49 years of age, 2 had lymphoma invading the retroperitoneal lymphatic system, as demonstrated by lymphangiography, and a 3rd patient had advanced ovarian carcinoma with metastasis to the mesenteric and retroperitoneal lymph nodes, diagnosed by laparotomy and confirmed at postmor-
880
MALAGELADA ETAL . TABLE
Patients
Vol.67,No. 5
1. Clinical and laboratory data of patients with cirrhosis
Age
Sex
Alkaline Tota l Albu- Triglyc- Cholesphospha- Prothromprotime min erides terol bin tase tein
Bilirubin
SGOT
mg/100 ml
KarmenU
Lowry units
sec over control
Bessey yr
g/ 100 ml
mg/ lOOml
Cirrhosis with chylous ascites 1 2 3
47 70 42
F M M
1.2 1.4 2.8
68 32 130
4.8 3.1 3.5
1.0 1.0 2.1
6.5 7.0 7.2
1.9 2.3 3.0
141 98 106
201 164 159
Cirrhosis with nonchylous ascites 1 2 3 4 5 6 7 8
42 66 56 52 64 45 56 62
M M F M M M M M
3.0 6.6 6.8 7.0 3.4 2.4 5.6 0.8
39 68 48 48 83 57 42 28
3.2 4.6 1.7 2.3 2.6 3.0 3.4 2.5
3.4 3.6 5.0. 3.0 4.0 2.5 3.7 0 .1
5.3 6.3 5.0 7.8 7.0 7.0 6.7 6.5
2.2 2.0 2.2 2.4 2.0 2.8 3.0 2.5
102 84 86
149 132 184
143 91 61
206 167 173
tern examination. The last patient had lymphatic obstruction due to metastatic seminoma which was confirmed by laparotomy. Methods Informed consent was obtained from all patients. After an overn ight fast, a paracentesis was performed using an intracath (needle size 17 gauge, catheter size 19 gauge) . The polyethylene catheter was left in place for 5 hr to collect samples of ascitic fluid. An intravenous infusion with 5% dextrose in water was maintained during the first 5 hr of the study in order to facilitate multiple blood collections. Base line samples of ascitic flu id and blood were taken, and a standard formula test meal (300 ml) was given orally to each patient. The test meal was composed of 20 mmoles of Triolein labeled with 50 !J.C of (3H ]oleic acid and 20 mmoles of Trioctanoin labeled with 50 !J.C of [14 C ]octanoic acid added to 270 ml of skim milk. (Triolein was purchased from Nutritional Biochemicals Co., Cleveland, Ohio. Trioctanoin was supplied by Eastman Organic Chemicals, Rochester, N . Y. ["H ]Oleic acid and ["C ]octanoic acid were obtained from the New England Nuclear Corp., Boston, Mass. The purity of the lipids was assessed by thin layer and gas liquid chromatography . The Triolein contained small amounts of contaminating long chain fatty acids.) The mixture was homogenized at room
temperature in a Waring Blendor and immediately fed to the patient. Samples of ascitic fluid (30 ml) and blood (15 ml) were obtained from all patients 30 min after ingestion of the test meal and every hour for the next 5 hr. Additional samples were taken at 8 hr from all patients of group 1, 4 patients of group 2, and 3 from group 3. Additional samples were obtained at 12, 24, 48, and 72 hr from all patients by repeated paracentesis and venipuncture. At each collection, the first 5 ml of ascitic fluid and the first 2 ml of blood were discarded. The abdominal wall was frequently massaged and the position of the patient changed to ensure mixing of the ascitic fluid . Ascitic fluid samples were stored in Erlenmeyer flasks at 4°C and analyzed within 12 hr. Blood was allowed to clot at 4°C, and the serum was then separated for additional analysis. Measurement of the ascitic fluid volume . The dilution method of Baker et al. 11 was used to measure the ascitic fluid volume with the following modifi'cations . Sulfobromophthalein standard solution (5%) was injected intraperitoneally just opposite to the catheter already in place to collect ascitic fluid . Approximately 2 mg of sulfobromophthalein were administered for each liter of estimated ascitic fluid volume. Sulfobromophthalein concentration in the ascitic fluid was determined by the method of Henry et al. 12 Analysis of ascitic fluid and blood samples. Oleic and octanoic acid were measured by a
November 1974
TABLE
Patients
Cirrhosis with nonchylous ascites 1 2 3 4 5 6 7 8 Mean ±
SE
Cirrhosis with chylous ascites 1 2 3 Mean ±
SE
Noncirrhotic patients with chylous ascites 1 2 3 4 Mean ±
SE
881
ORIGIN OF FAT IN CHYLOUS ASCITES 2. Volume and composition of ascitic fluid
Ascites volume
Protein
Total fatty acids"
liters
g/100 ml
9.1 4.1 8.1 13.6 6.4 7.8 8.3 6.8
1.0 2.1 0.5 1.0 0.2 1.0 0.9 3.0
80 104 92 157 150 104 50 96
7.9 ± 0.9
1.2 ± 0.3
104 ± 12
9.8 12.1 6.1
0.9 2.6 0.6
226 529 470
9.3 ± 1.7
1.4 ± 0.6
9.1 6.8 7.3 7.2
1.0 2.1 2.8 3.8
7.6 ± 0 .5
2.4 ± 0.6
Triglycerides Phospholipids
Cholesterol
mg/lOOml
30 32 20 42 54 29 18 37
40 51 40 36 48 37 20 32
23 ± 4
37 ± 3
150 400 335
50 83 80
26 46 55
408 ± 92
295 ± 74
71 ± 10
42 ± 8
772 940 1200 1558
626 717 991 1260
76 129 122 180
70 94 87 113
898 ± 143
126 ± 2
92 ± 9
1117±171
10 21 32 79 48 38 12 27 37 ± 9
" Esterified and unesterified fatty acids.
previously described method . 13 Radioactivity was determined in a liquid scintillation counter (model LS-250, Beckman Instruments , Inc., Fullerton, Calif.) using Bray's scintillator solution . 14 The chylomicrons of ascitic fluid and serum (Sf value greater than 400) were isolated by the method of Alaupovic et al. 15 Isolated chylomicron fraction (1 ml) and the non-chylomicron fraction (1 ml) were extracted and the radioactivity was measured. Triglycerides, phospholipids, and cholesterol were measured in the ascitic fluid and serum of the fasting patients following standard methods.' 6 - 18 Thin layer chromatography of the ascitic fluid was performed in samples obtained 5 and 12 hr after test meal ingestion (all of group 1, 5 from group 2, and 3 from group 3) . Aliquots ( 1 ml) of each of the followingwhole ascitic fluid, the chylomicron fraction,
and the chylomicron-free fraction-were extracted by the method of Dole and Meinertz. 19 Two milliliters of the upper phase were lyophilized and redissolved in 20 ttl of chloroform. Ten ttl of this solution were applied to a thin layer plate coated with Adsorbosil-5 (250 tt thick) (Applied Science Laboratories, State College, Pa.) and developed for 20 em in the solvent system: petroleum ether-diethyl ether-glacial acetic acid (90:10:1, by volume). The spots were identified by reference standard solutions after staining with iodine vapor. (Radioactivity was too weak for autoradiography. Increasing the radioactivity in the test meals would have exceeded the safety standards for human experiments.) Similar lipid analysis was carried out in ~:>amples of serum obtained from the same patients, 5 hr after ingestion of the test meal. Student's t-test was used for statistical comparison between groups.
882
MALAGELADA ET AL.
Results Analy sis of the Lipid Fractions of the Ascitic Fluid and Serum Cirrhotic patients with chylous ascites had a 4-fold higher total fat concentration in ascitic fluid when compared with cirrhotic patients with nonchylous ascites (table 2). The relative concentration in ascites of the three constituents of the chylomicron lipid fraction (triglycerides, phospholipids, and cholesterol) are expressed in figure 1 on triangular coordinates. Patients with chylous ascites are located in the lower left angle of the triangle indicating a high ratio of triglycerides to phospholipids and cholesterol, as compared with patients with nonchylous ascites. The lipid composition of chylous ascites is very similar to that of human thoracic duct lymph after ingestion of a fat meal as determined by Bartlett et al. 20 (triangular point in fig. 1). Thin layer chromatography of the chylomicron fraction of ascitic fluid and serum revealed all fatty acid label to be present in esterified form . The fraction of medium chain fatty acids incorporated as triglycerides in the ascitic chylomicrons was approximately 6% of long chain triglycerides. 100
Vol . 67, No .5
This is in agreement with the observations by Lee et al. 21 who demonstrated increased lymphatic transport of medium chain triglycerides when administered in combination with long chain triglycerides. Kinetics of [3H]Oleic Acid Given as Triolein In ascites . More than 80% of the total ascites fluid [3 H ]oleic acid was recovered as triglyceride in ascites chylomicrons, in contrast to less than half in clear ascites (fig. 2). Since these ratios did not change throughout the observation periods, the fractions of 3 H-lipid in and outside the chylomicrons remained constant. The total amounts of [3H]chylornicron triglyceride in ascites at various times after ingestion of the labeled test meal during a 72-hr observation period are shown in figure 3. It seems reasonable to assume that these curves also represent the kinetics of 3 H-labeled chylomicrons in ascites. The time of appearance (1 hr) and the time interval from ingestion to the peak levels (5 hr) were similar in the three groups. The disappearance rate after 12 hr was also similar (average half-life time 58 hr), and therefore the final decay rate of the chylo0
20
BO
40 CHOLESTEROL,%
PHOSPHOLIPIDS,%
•,/
40
. •' -------------------·--·<,
60
'
''
20
''
\
80
''
''
''
0~----------------~----'~'--.--.~~100 100
80
60
40
20
0
TRIGLYCERIDE$ , %
FIG. 1. Lipid composition of the ascitic fluid. Concentration (mg/100 ml) ratios for triglycerides, phospholipids, and cholesterol expressed in triangular coordinates. e, patients with cirrhosis and nonchylous ascites; 0, patients with cirrhosis and chylous ascites; x , noncirrhotic patients with chylous ascites; .&, lipid composition of human thoracic; duct lymph after ingestion of a fat meal. 20
November 1974
ORIGIN OF FAT IN CHYLOUS ASCITES 100
883 I Mean ± SE
% 3 H- Labelled
60 Li pid in lhe C h ylomicron Fraclion 40 of Asc i les
;A-I
;r
T
20
00
12
6
48
24
Tesi 1 Meal
72
Hours
FIG. 2. Percentage of total 'H-lipid in ascites incorporated in the chylomicron fraction . All ['H ]oleic acid in chylomicrons was present in esterified form as ['H]chylomicron t ri glyceride. •--e, patients with cirrhosis and non chylous ascites; 0--0, patients with cirrhosis and chylous ascites; x - -x, noncirrhotic patients with chylous ascites.
10 5
I Mean ±S.E.
I
!;_ '/ __~------?----~ ~ ~ -~ .
~i
10 3 3H Oleic Acid in Chylomicron Trig I yceride
(pmolesl
10 2
I "l 0 Test •Mea l
6
12
48
24
72
H ours
FIG. 3. Total amounts of ['H]oleic acid in chylomicron triglyceride in ascites. •--e, patients with cirrhosis and nonchylous ascites; 0--0, patients with cirrhos is and chylous ascites; x - - x , noncirrhotic patients with chylous ascites.
884
Vol.67,No .5
MALAGELADA ET AL.
microns was equal in patients with clear and chylous ascites. There was, however, a marked difference in the peak accumulation (at 5 hr) of ingested lipids in the ascitic fluid. Only 0.6% of the administered dose of Triolein was present in ascitic fluid during peak levels in cirrhotic patients with clear ascites versus 8% in cirrhotic patients with chylous ascites. The 4 noncirrhotic patients with lymphatic obstruction accumulated, on the average, a peak value of 22% of the ingested dose. Each group was significantly different from the other two (P < 0.01). In serum. In contrast to ascites, the ratio between [3H ]oleic acid incorporated in serum chylomicrons and in the nonchylomicron fraction was not constant, but increased gradually with the accumulation of chylomicrons in blood. At peak levels (4 to 5 hr), the chylomicron fraction accounted for 32% of the total 3H-radioactivity in serum, declining to less than 6% within 12 hr. Appearance, peak levels, and final decay rates of [3H ]chylomicron triglyceride in serum were similar in the three groups of patients . Peak levels were observed between 4 and 5 hr. The half-life time was much shorter than in ascites, namely, 1.8 hr. Also, no significant differences were de600
tected between the three groups with regard to total 3 H-lipid concentrations in serum.
C]Octanoic Acid Given as Trioctanoin In ascites. [14C ]Octanoic acid appeared in ascites in two forms with different kinetics: (1) in esterified form incorporated in the chylomicrons, and (2) as free fatty acid. 1. Patients with clear ascites had little 14 [ C ]octanoic acid incorporated in the chylomicrons (fig. 4A) . The amounts of 14 [ C]chylomicron triglyceride in chylous ascites were proportional to total fat concentration of the ascitic fluid . Final decay rate of [14C]triglyceride in the chylomicrons was much slower than the free acid and comparable to [3H ]chylomicron triglyceride. 2. Within 5 hr after administration of the test meal, free octanoic acid accumulated rapidly in the ascites of both cirrhotic groups reaching peak concentrations of approximately the same magnitude (fig. 4B). This was followed by a rapid decline with small quantities remaining after 12 hr. Much smaller amounts entered the ascitic fluid of the noncirrhotic subjects, amounting to less than 20% of the peak value in the cirrhotic subjects. [
14
B
A
400
JJ moles
---------X ---------X 0~~~==-.---------L------~
0
1
6
Test Meal
12
24
48 Hours
72
0
6
1 Te st Meal
12
24
48
72
Hours
FIG. 4. Total amounts of ["C )octanoic acid in ascites . A, the left side of the figure shows the fraction of ["C]octanoic acid incorporated in the ascitic chylomicrons as ["C]chylomicron triglyceride (micromoles). B, the right side represents the fraction of [14C]octanoic acid not incorporated in the chylomicrons, present as unesterified fatty acid (micromoles). e-e, patients with cirrhosis and nonchylous ascites; 0 - 0 , patients with cirrhosis and chylous ascites; x x, noncirrhotic patients with chylous ascites.
November 1974
ORIGIN OF FAT IN CHYLOUS ASCITES
In serum. Peak serum octanoic levels occurred between 30 and 60 min after ingestion. As it has been noted before, cirrhotic patients have much higher concentrations than noncirrhotic subjects (peak serum levels: 457 ± 44 J.LM, mean ± SE versus 163 ± 19 J.LM, respectively, P < 0.01) due to shunting and impaired parenchymal function 13 with slower rate of disappearance (5-hr concentration: 94 ± 11 J.LM versus 12 ± 5 J.LM, respectively, P < 0.01) . In all patients, only a small percentage (0 to 6%) of [14 C ]octanoic acid was present in the chylomicron fraction. Discussion This study has demonstrated that most of the ingested triglycerides recovered from chylous ascites were present in the chylomicrons. In the cirrhotic patients, activity of labeled long chain fat found in chylous ascites was much greater (8% of the ingested radioactivity) than in clear ascites (0.6%). An even greater proportion was recovered from that of the noncirrhotic patients (22%) in whom chylous ascites consisted most likely of intestinal lymph. Incorporation of [3H ]oleic acid into ascitic chylomicrons as [3H]chylomicron triglyceride (fig. 2) was significantly higher in the subjects with chylous ascites than in those with clear ascites (P < 0.01). This suggests that intestinal lymph was the main source of chylomicrons in chylous ascites as chylomicrons can permeate intestinal lymphatic capillaries. 22 Chylomicron removal from ascitic fluid was about 30 times slower than removal from blood, as indicated by the respective final decay rates (half-life time: 58 versus 1.8 hr). Nevertheless, it appears that the absolute removal rate from ascites (micromoles per hour) was at least partially dependent on concentrations of chylomicrons in ascitic fluid. Our data also show that the rate of entry of chylomicrons into the ascites was different in three groups, while the removal rate was uninfluenced by disease process (final decay rates were similar), suggesting that the faster rate of appearance and high concentrations of chylomicrons found in chylous ascites were due to a greater leakage of chylomicrons
885
from intestinal lymph into the peritoneal cavity. Removal of the chylomicrons from ascitic fluid by centrifugation completely eliminated the chylous quality of our samples in all instances. The experiments with medium chain fatty acid ( [14 C ]octanoic acid) were designed to study transfer of water-soluble lipid from the vascular compartment into ascites. The absorbed medium chain fatty acid, transported mainly via the portal venous system as free fatty acid, rapidly accumulated in ascitic fluid of both cirrhotic groups. Previous studies have shown that at the concentrations of octanoic acid measured in blood of the cirrhotic patients, a significant portion is free and not bound to protein. 23 Non-protein-bound octanoate, which is water- and fat-soluble, probably diffuses through many membrane systems. It has been shown previously that the non-protein-bound serum fraction of octanoate in the cirrhotic patients is 4 to 5 times higher than normal. Spinal fluid levels were linearly related with, and of the same magnitude of, the free serum octanoate concentration. 23 The same mechanism of rapid diffusion of the non-protein-bound fatty acids from blood into ascites can probably explain the ascitic fluid levels since, in both cirrhotic groups, the concentrations of octanoate not incorporated into the chylomicrons were also 5 times higher as compared with the noncirrhotic patients (fig. 4B). Not all medium chain lipids are transported via the lymphatics as shown by the levels of octanoic acid incorporated in the chylomicrons in ascitic fluid as 14 [ C]chylomicron triglyceride (see fig . 4A), with final decay rates being similar to those of 3H (long chain) chylomicron triglyceride . These results further support the lymphatic origin of chylomicrons in ascitic fluid and suggest that chylous ascites in the cirrhotic originates both from lymph and blood with the chylomicron lipid arising from intestinal lymph and the free fatty acids from blood. Analysis of the composition of chylomicron lipid might provide additional information on the possible mechanism of chylous ascites. Experiments by Fraser 24 have
886
MALAGELADA ET AL.
demonstrated that the size of chylomicrons in lymph is related to the ratios between triglycerides, phospholipids, and cholesterol. When the three main lipid components of ascites are plotted in a triangular system of coordinate (fig. 1), the proportions of the three lipids in cirrhotic patients with chylous ascites appear to be similar to the lymphatic ascites of the noncirrhotic patients (and identical to intestinal lymph), which indicates that the size of the chylomicrons is probably equal. The composition of the lipid fractions in clear ascites, however, is significantly different from chylous ascites, being in the range of the much smaller chylomicrons. As patients with cirrhosis have usually an elevated lymphatic pressure, 25 it is suggested that in all cirrhotic patients with ascites, small chylomicrons escape continuously from lymph capillaries into the ascitic fluid. Chylous ascites in the cirrhotic patient may result from exceptionally high lymphatic pressure leading to rupture of lymph vessels and leakage of whole intestinal lymph into the peritoneal cavity. This assumption is consistent with the anatomical finding of subserosa! intestinallymphagiectasis, recently reported in patients with liver cirrhosis and chylous ascites. 26 REFERENCES 1. Malagelada JR, Iber FL, Linscheer WG: The origin of fat in chylous ascites of patients with liver cirrhosis (abstr). Gastroenterology 62:780, 1972 2. Nix JT, Albert M, Dugas JE, eta! : Chylothorax and chylous ascites: a study of 302 selected cases. Am J Gastroenterol 28 :40-55, 1957 3. Lamotte M, Lamotte-Barrilon, S. Guerre J: L'ascite chyliforme des cirrhotiques. Arch Fr Mal App Dig 57:447-458, 1968 4. Kelley ML, Butt HR: Chylous ascites: an analysis of its etiology. Gastroenterology 39:161-170, 1960 5. Kelley ML, Logan VW: The problem of milky ascites. NY State J Med 61:2307-2315, 1961 6. Waterhouse C, Jaenike JR, Marinetti G: Studies on the nature and origin of pseudochylous ascites. Trans Assoc Am Physicians 71:312-321, 1958 7. Liebowitz HR: Pathogenesis of ascites in cirrhosis of the liver. NY State J Med 69:1895- 2024, 1969 8. Albrink MJ, Glenn WWL, Peters JP, eta!: The transport of lipids in chyle. J Clin . Invest 34:1467-1475, 1955
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9. Morris B, Courtice FC: The origin of chylomicrons in the cervical and hepatic lymph. Q J Exp Physiol 41:341- 348, 1956 10. Courtice FC: The origin of lipoproteins in lymph. In The Lymph and Lymphatic System. Edited by HS Mayerson. Springfield, CC Thomas, 1968, p 119 11. Baker L, Puestow RC, Kruger S, eta!: Estimation of ascitic fluid percent volumes. J Lab Clin Med 39:30-36, 1952 12. Henry RJ, Chiamori N, Ware AG: Determination of bromsulfonphthalein in serum by means of acetone precipitation of proteins. Am J Clin Pathol 32:201- 227, 1959 13. Linscheer WG, Castell DO, Platt RR: A new method for evaluation of portasystemic shunting. The rectal octanoate tolerance test. Gastroenterology 57:415-423, 1969 14. Bray GA: A simple efficient lipid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal Biochem 1:279-285, 1960 15. Alaupovic P, Furman RH, Failor WH, et al: Isolation and characterization of human chyle chylomicrons and lipoproteins. Ann NY Acad Sc 149:791- 807, 1968 16. Noble RP, Campbell FM: Improved accuracy in automated fluorometric determination of plasma triglycerides. Clin Chern 16:166- 170, 1970 17. Henry RJ: Clinical Chemistry. New York, Harper and Row, 1965, p 841-843 18. Total cholesterol procedure (N-24). AutoAnalyzer Manual. Tarrytown, N.Y., 1965 19. Dole VP, Meinertz H: Microdeterminationoflong chain fatty acids in plasma and tissues. J Biol Chern 235:2595-2597, 1960 20. Bartlett RH, Failor WH, Zarafonetis CJD: Lipid studies on human lymph and chyle. JAMA 187:126-127, 1964 21. Lee DS , Hashim SA, Van Itallie TB: Effect of long chain triglyceride on chylous transport of medium chain fatty acids. Am J Physiol 214:294- 297, 1968 22. Courtice FC: The origin of lipoproteins in lymph. In The Lymph and Lymphatic System. Edited by HS Mayerson. Springfield, CC Thomas, 1968, p 98 23. Linscheer WG, Blum AL, Platt RR: Transfer of medium chain fatty acids from blood to spinal fluid in patients with cirrhosis. Gastroenterology 58:509-515, 1970 24. Fraser R: Size and lipid composition of chylomicrons of different Svedberg units of flotation. J Lipid Res 11:60-65, 1970 25. Dumont AE, Mulholland JH: Alterations in thoracic duct lymph flow in hepatic cirrhosis. Ann Surg 156:668- 677, 1962. 26. Caroli J, Ouahnich M: Ascite Chyleuse du cirrhotique. Rev Med Chir Mal Foie 44:99-108, 1969
GASTROENTEROLOGY" 67:878-886, 1974
Copyright © 1974 by The Williams & Wilkins Co.
Printed in U .S .A.
ORIGIN OF FAT IN CHYLOUS ASCITES OF PATIENTS WITH LIVER CIRRHOSIS JUAN
R.
MALAGELADA, FRANK
L.
lBER, AND WILLEM
G.
LINSCHEER
Lemuel Shattuck Hospital, Department of Medicine, Tufts University School of Medicine , Boston, Massachus etts
To investigate the origin of fat in chylous ascites of patients with liver cirrhosis, labeled medium and long chain fatty acids ( [14 C ]octanoic and [3H]oleic acid, respectively) were fed to 3 cirrhotic patients with chylous ascites as well as to two control groups: patients with cirrhosis and clear ascites and noncirrhotic subjects with chylous ascites. The time of appearance, time interval from ingestion to peak levels, and half-life time of decay of labeled long chain fatty acids in ascitic chylomicrons were similar in all groups of patients. In the cirrhotic patients, the peak activity of long chain fat found in chylous ascites (8% of ingested radioactivity) was much greater than in clear ascites (0.6%). An even greater amount of ingested long chain fat was found in the chylous ascites of noncirrhotic patients (22%). More than 80% of the ingested [3 H ]oleic acid present in chylous ascites was incorporated in esterified form in chylomicrons as compared to less than 50% in clear ascitic fluid. This significantly higher (P < 0.01) incorporation of 3H radioactivity in subjects with chylous ascites when serum levels were identical suggests that intestinal lymph was the main source of ascitic chylomicrons. The appearance, concentration, and decay rate in ascites of [14 C ]octanoic acid, not incorporated in the chylomicrons, was similar in cirrhotic patients with clear or chylous ascitic fluid, suggesting a similar pathway of transport of the nonprotein-bound [14 C )octanoic acid. Analysis of the relative concentrations of triglycerides, phospholipids, and cholesterol in ascitic fluid revealed a relatively low proportion of triglycerides in clear ascitic fluid which indicates that most of the triglycerides are present in the smaller particles, probably a combination of small chylomicrons, very low, low, and high density lipoproteins. It can be concluded from our studies that only small chylomicrons, probably leaking from the intestinal lymphatics, enter the ascites of cirrhotic patients with clear ascitic fluid. When the number and size of these chylomicrons increases, the ascitic fluid acquires a chylous nature. Whether the underlying pathogenesis involves abnormal permeability or rupture of the lymphatics is unknown, but the process may reverse with improvement of liver function. Chylous ascites is an infrequent feature of decompensated cirrhosis of the liver. 2 - 5
The origin of fat in ascitic fluid of these patients is unclear. 6 Ascites in cirrhosis is
Received March 2, 1973. Accepted April17, 1974. This work has been reported in part. 1 Address requests for reprints to: Dr. Willem G.
Linscheer, Department of Gastroenterology, Veterans Administration Hospital, Irving Avenue and University Place, Syracuse, New York 13210.
878
November 1974
879
ORIGIN OF FAT IN CHYLOUS ASCITES
thought to be produced by a combination of filtration of fluid through the splanchnic capillary system as a result of portal hypertension and decreases osmotic pressure (low albumin), leakage of lymph from the liver surface (weeping liver), and possibly also from the intestinal lymphatic system. 7 The milky appearance of chylous ascites is thought to be due to the high concentration of chylomicrons. 8 As hepatic lymph contains only a low concentration of chylomicrons, 9 and splanchnic venous capillaries are considered to be impermeable for chylomicrons, 10 the most likely source of chylomicrons in ascites is the intestinal lymph. Although rupture of peritoneal lymph vessel has been demonstrated in patients with lymphatic obstruction due to malignancy, it has seldom been reported in patients with cirrhosis and chylous ascites. This study was designed to investigate the origin of lipids in chylous ascites of cirrhotic patients. As triglycerides are the main constituents of chylomicrons, we have studied the appearance and turnover of ingested 3 H-labeled long chain triglycerides in the ascitic fluid chylomicron fraction. 14 C-labeled medium chain triglycerides, given simultaneously, were used to study transfer of water-soluble soaps from portal and peripheral blood into ascites. Patients with chylous ascites associated with cirrhosis were compared with a group of patients with clear ascites associated with cirrhosis and with a third group of patients with chylous ascites due to lymphatic obstruction. This work was supported in part by Research Grant AM 11566 and Training Grant AM 05424 from the National Institutes of Health, U.S. Public Health Service, and a grant from The John A. Hartford Foundation. Dr. Juan R. Malagelada was a recipient of a Juan March Foundation Fellowship. His present address is: Department of Internal Medicine and Gastroenterology, Mayo Clinic, Rochester, Minnesota 55901. The authors are indebted to Drs. Hyman J . Zimmerman, Leo Stolbach, and Marvin Krant for permission to study patients under their care; to Miss Pat Burns and Miss Sheila Lieberman for technical assistance; and to Dr. Neville Hoffman for his critical review of the manuscript.
Experimental Procedure Patients Fifteen patients divided into three groups were studied (tables 1 and 2). Group 1: cirrhotic patients with chylous ascites (3 patients). All 3 patients were chronic alcoholics in whom the diagnosis of cirrhosis of the liver was established by biopsy. Patient 1, female , 47 years old . One year prior to the study she developed symptoms of cirrhosis with ascites. Her symptoms did not respond to treatment, including diuretics, and she underwent periodic paracentesis to relieve intraabdominal pressure. The ascitic fluid was clear until 10 weeks prior to study when it became progressively milky. No signs of malignancy or mechanical obstruction of the lymphatic system were found at autopsy 2 months after the study. Patient 2, male, 70 years old. Clinical symptoms of cirrhosis appeared 5 years before study; ascites had been present for 18 months . Paracentesis 3 months prior to study revealed chylous ascitic fluid and an abdominallymphangiogram performed at this time showed no abnormality. He died of myocardial infarction 3 months after study. Permission for autopsy was not obtained. Patient 3, male, 42 years old. \1anifestations of cirrhosis with ascites developed at least 8 months before study, but chylous ascites was noted only for the last month. After the study he responded favorably to abstinence froni alcohol, bed rest, and a nutritious diet, and his ascites disappeared completely. He has been followed closely for 1 year and no recurrence of the ascites has been observed. Clinical and laboratory findings at present are consistent with compensated cirrhosis. Group 2: cirrhotic patients with clear ascites (8 patients). The 8 patients, averaging 53 years of age, were all chronic alcoholics in whom cirrhosis was confirmed by liver biopsy, and all had had ascites for a minimum period of 6 weeks. Group 3: noncirrhotic patients with intraabdominal malignancies and chylous ascites (4 patients: two females and two males). Of the 4 patients, who averaged 49 years of age, 2 had lymphoma invading the retroperitoneal lymphatic system, as demonstrated by lymphangiography, and a 3rd patient had advanced ovarian carcinoma with metastasis to the mesenteric and retroperitoneal lymph nodes, diagnosed by laparotomy and confirmed at postmor-
880
MALAGELADA ETAL . TABLE
Patients
Vol.67,No. 5
1. Clinical and laboratory data of patients with cirrhosis
Age
Sex
Alkaline Tota l Albu- Triglyc- Cholesphospha- Prothromprotime min erides terol bin tase tein
Bilirubin
SGOT
mg/100 ml
KarmenU
Lowry units
sec over control
Bessey yr
g/ 100 ml
mg/ lOOml
Cirrhosis with chylous ascites 1 2 3
47 70 42
F M M
1.2 1.4 2.8
68 32 130
4.8 3.1 3.5
1.0 1.0 2.1
6.5 7.0 7.2
1.9 2.3 3.0
141 98 106
201 164 159
Cirrhosis with nonchylous ascites 1 2 3 4 5 6 7 8
42 66 56 52 64 45 56 62
M M F M M M M M
3.0 6.6 6.8 7.0 3.4 2.4 5.6 0.8
39 68 48 48 83 57 42 28
3.2 4.6 1.7 2.3 2.6 3.0 3.4 2.5
3.4 3.6 5.0. 3.0 4.0 2.5 3.7 0 .1
5.3 6.3 5.0 7.8 7.0 7.0 6.7 6.5
2.2 2.0 2.2 2.4 2.0 2.8 3.0 2.5
102 84 86
149 132 184
143 91 61
206 167 173
tern examination. The last patient had lymphatic obstruction due to metastatic seminoma which was confirmed by laparotomy. Methods Informed consent was obtained from all patients. After an overn ight fast, a paracentesis was performed using an intracath (needle size 17 gauge, catheter size 19 gauge) . The polyethylene catheter was left in place for 5 hr to collect samples of ascitic fluid. An intravenous infusion with 5% dextrose in water was maintained during the first 5 hr of the study in order to facilitate multiple blood collections. Base line samples of ascitic flu id and blood were taken, and a standard formula test meal (300 ml) was given orally to each patient. The test meal was composed of 20 mmoles of Triolein labeled with 50 !J.C of (3H ]oleic acid and 20 mmoles of Trioctanoin labeled with 50 !J.C of [14 C ]octanoic acid added to 270 ml of skim milk. (Triolein was purchased from Nutritional Biochemicals Co., Cleveland, Ohio. Trioctanoin was supplied by Eastman Organic Chemicals, Rochester, N . Y. ["H ]Oleic acid and ["C ]octanoic acid were obtained from the New England Nuclear Corp., Boston, Mass. The purity of the lipids was assessed by thin layer and gas liquid chromatography . The Triolein contained small amounts of contaminating long chain fatty acids.) The mixture was homogenized at room
temperature in a Waring Blendor and immediately fed to the patient. Samples of ascitic fluid (30 ml) and blood (15 ml) were obtained from all patients 30 min after ingestion of the test meal and every hour for the next 5 hr. Additional samples were taken at 8 hr from all patients of group 1, 4 patients of group 2, and 3 from group 3. Additional samples were obtained at 12, 24, 48, and 72 hr from all patients by repeated paracentesis and venipuncture. At each collection, the first 5 ml of ascitic fluid and the first 2 ml of blood were discarded. The abdominal wall was frequently massaged and the position of the patient changed to ensure mixing of the ascitic fluid . Ascitic fluid samples were stored in Erlenmeyer flasks at 4°C and analyzed within 12 hr. Blood was allowed to clot at 4°C, and the serum was then separated for additional analysis. Measurement of the ascitic fluid volume . The dilution method of Baker et al. 11 was used to measure the ascitic fluid volume with the following modifi'cations . Sulfobromophthalein standard solution (5%) was injected intraperitoneally just opposite to the catheter already in place to collect ascitic fluid . Approximately 2 mg of sulfobromophthalein were administered for each liter of estimated ascitic fluid volume. Sulfobromophthalein concentration in the ascitic fluid was determined by the method of Henry et al. 12 Analysis of ascitic fluid and blood samples. Oleic and octanoic acid were measured by a
November 1974
TABLE
Patients
Cirrhosis with nonchylous ascites 1 2 3 4 5 6 7 8 Mean ±
SE
Cirrhosis with chylous ascites 1 2 3 Mean ±
SE
Noncirrhotic patients with chylous ascites 1 2 3 4 Mean ±
SE
881
ORIGIN OF FAT IN CHYLOUS ASCITES 2. Volume and composition of ascitic fluid
Ascites volume
Protein
Total fatty acids"
liters
g/100 ml
9.1 4.1 8.1 13.6 6.4 7.8 8.3 6.8
1.0 2.1 0.5 1.0 0.2 1.0 0.9 3.0
80 104 92 157 150 104 50 96
7.9 ± 0.9
1.2 ± 0.3
104 ± 12
9.8 12.1 6.1
0.9 2.6 0.6
226 529 470
9.3 ± 1.7
1.4 ± 0.6
9.1 6.8 7.3 7.2
1.0 2.1 2.8 3.8
7.6 ± 0 .5
2.4 ± 0.6
Triglycerides Phospholipids
Cholesterol
mg/lOOml
30 32 20 42 54 29 18 37
40 51 40 36 48 37 20 32
23 ± 4
37 ± 3
150 400 335
50 83 80
26 46 55
408 ± 92
295 ± 74
71 ± 10
42 ± 8
772 940 1200 1558
626 717 991 1260
76 129 122 180
70 94 87 113
898 ± 143
126 ± 2
92 ± 9
1117±171
10 21 32 79 48 38 12 27 37 ± 9
" Esterified and unesterified fatty acids.
previously described method . 13 Radioactivity was determined in a liquid scintillation counter (model LS-250, Beckman Instruments , Inc., Fullerton, Calif.) using Bray's scintillator solution . 14 The chylomicrons of ascitic fluid and serum (Sf value greater than 400) were isolated by the method of Alaupovic et al. 15 Isolated chylomicron fraction (1 ml) and the non-chylomicron fraction (1 ml) were extracted and the radioactivity was measured. Triglycerides, phospholipids, and cholesterol were measured in the ascitic fluid and serum of the fasting patients following standard methods.' 6 - 18 Thin layer chromatography of the ascitic fluid was performed in samples obtained 5 and 12 hr after test meal ingestion (all of group 1, 5 from group 2, and 3 from group 3) . Aliquots ( 1 ml) of each of the followingwhole ascitic fluid, the chylomicron fraction,
and the chylomicron-free fraction-were extracted by the method of Dole and Meinertz. 19 Two milliliters of the upper phase were lyophilized and redissolved in 20 ttl of chloroform. Ten ttl of this solution were applied to a thin layer plate coated with Adsorbosil-5 (250 tt thick) (Applied Science Laboratories, State College, Pa.) and developed for 20 em in the solvent system: petroleum ether-diethyl ether-glacial acetic acid (90:10:1, by volume). The spots were identified by reference standard solutions after staining with iodine vapor. (Radioactivity was too weak for autoradiography. Increasing the radioactivity in the test meals would have exceeded the safety standards for human experiments.) Similar lipid analysis was carried out in ~:>amples of serum obtained from the same patients, 5 hr after ingestion of the test meal. Student's t-test was used for statistical comparison between groups.
882
MALAGELADA ET AL.
Results Analy sis of the Lipid Fractions of the Ascitic Fluid and Serum Cirrhotic patients with chylous ascites had a 4-fold higher total fat concentration in ascitic fluid when compared with cirrhotic patients with nonchylous ascites (table 2). The relative concentration in ascites of the three constituents of the chylomicron lipid fraction (triglycerides, phospholipids, and cholesterol) are expressed in figure 1 on triangular coordinates. Patients with chylous ascites are located in the lower left angle of the triangle indicating a high ratio of triglycerides to phospholipids and cholesterol, as compared with patients with nonchylous ascites. The lipid composition of chylous ascites is very similar to that of human thoracic duct lymph after ingestion of a fat meal as determined by Bartlett et al. 20 (triangular point in fig. 1). Thin layer chromatography of the chylomicron fraction of ascitic fluid and serum revealed all fatty acid label to be present in esterified form . The fraction of medium chain fatty acids incorporated as triglycerides in the ascitic chylomicrons was approximately 6% of long chain triglycerides. 100
Vol . 67, No .5
This is in agreement with the observations by Lee et al. 21 who demonstrated increased lymphatic transport of medium chain triglycerides when administered in combination with long chain triglycerides. Kinetics of [3H]Oleic Acid Given as Triolein In ascites . More than 80% of the total ascites fluid [3 H ]oleic acid was recovered as triglyceride in ascites chylomicrons, in contrast to less than half in clear ascites (fig. 2). Since these ratios did not change throughout the observation periods, the fractions of 3 H-lipid in and outside the chylomicrons remained constant. The total amounts of [3H]chylornicron triglyceride in ascites at various times after ingestion of the labeled test meal during a 72-hr observation period are shown in figure 3. It seems reasonable to assume that these curves also represent the kinetics of 3 H-labeled chylomicrons in ascites. The time of appearance (1 hr) and the time interval from ingestion to the peak levels (5 hr) were similar in the three groups. The disappearance rate after 12 hr was also similar (average half-life time 58 hr), and therefore the final decay rate of the chylo0
20
BO
40 CHOLESTEROL,%
PHOSPHOLIPIDS,%
•,/
40
. •' -------------------·--·<,
60
'
''
20
''
\
80
''
''
''
0~----------------~----'~'--.--.~~100 100
80
60
40
20
0
TRIGLYCERIDE$ , %
FIG. 1. Lipid composition of the ascitic fluid. Concentration (mg/100 ml) ratios for triglycerides, phospholipids, and cholesterol expressed in triangular coordinates. e, patients with cirrhosis and nonchylous ascites; 0, patients with cirrhosis and chylous ascites; x , noncirrhotic patients with chylous ascites; .&, lipid composition of human thoracic; duct lymph after ingestion of a fat meal. 20
November 1974
ORIGIN OF FAT IN CHYLOUS ASCITES 100
883 I Mean ± SE
% 3 H- Labelled
60 Li pid in lhe C h ylomicron Fraclion 40 of Asc i les
;A-I
;r
T
20
00
12
6
48
24
Tesi 1 Meal
72
Hours
FIG. 2. Percentage of total 'H-lipid in ascites incorporated in the chylomicron fraction . All ['H ]oleic acid in chylomicrons was present in esterified form as ['H]chylomicron t ri glyceride. •--e, patients with cirrhosis and non chylous ascites; 0--0, patients with cirrhosis and chylous ascites; x - -x, noncirrhotic patients with chylous ascites.
10 5
I Mean ±S.E.
I
!;_ '/ __~------?----~ ~ ~ -~ .
~i
10 3 3H Oleic Acid in Chylomicron Trig I yceride
(pmolesl
10 2
I "l 0 Test •Mea l
6
12
48
24
72
H ours
FIG. 3. Total amounts of ['H]oleic acid in chylomicron triglyceride in ascites. •--e, patients with cirrhosis and nonchylous ascites; 0--0, patients with cirrhos is and chylous ascites; x - - x , noncirrhotic patients with chylous ascites.
884
Vol.67,No .5
MALAGELADA ET AL.
microns was equal in patients with clear and chylous ascites. There was, however, a marked difference in the peak accumulation (at 5 hr) of ingested lipids in the ascitic fluid. Only 0.6% of the administered dose of Triolein was present in ascitic fluid during peak levels in cirrhotic patients with clear ascites versus 8% in cirrhotic patients with chylous ascites. The 4 noncirrhotic patients with lymphatic obstruction accumulated, on the average, a peak value of 22% of the ingested dose. Each group was significantly different from the other two (P < 0.01). In serum. In contrast to ascites, the ratio between [3H ]oleic acid incorporated in serum chylomicrons and in the nonchylomicron fraction was not constant, but increased gradually with the accumulation of chylomicrons in blood. At peak levels (4 to 5 hr), the chylomicron fraction accounted for 32% of the total 3H-radioactivity in serum, declining to less than 6% within 12 hr. Appearance, peak levels, and final decay rates of [3H ]chylomicron triglyceride in serum were similar in the three groups of patients . Peak levels were observed between 4 and 5 hr. The half-life time was much shorter than in ascites, namely, 1.8 hr. Also, no significant differences were de600
tected between the three groups with regard to total 3 H-lipid concentrations in serum.
C]Octanoic Acid Given as Trioctanoin In ascites. [14C ]Octanoic acid appeared in ascites in two forms with different kinetics: (1) in esterified form incorporated in the chylomicrons, and (2) as free fatty acid. 1. Patients with clear ascites had little 14 [ C ]octanoic acid incorporated in the chylomicrons (fig. 4A) . The amounts of 14 [ C]chylomicron triglyceride in chylous ascites were proportional to total fat concentration of the ascitic fluid . Final decay rate of [14C]triglyceride in the chylomicrons was much slower than the free acid and comparable to [3H ]chylomicron triglyceride. 2. Within 5 hr after administration of the test meal, free octanoic acid accumulated rapidly in the ascites of both cirrhotic groups reaching peak concentrations of approximately the same magnitude (fig. 4B). This was followed by a rapid decline with small quantities remaining after 12 hr. Much smaller amounts entered the ascitic fluid of the noncirrhotic subjects, amounting to less than 20% of the peak value in the cirrhotic subjects. [
14
B
A
400
JJ moles
---------X ---------X 0~~~==-.---------L------~
0
1
6
Test Meal
12
24
48 Hours
72
0
6
1 Te st Meal
12
24
48
72
Hours
FIG. 4. Total amounts of ["C )octanoic acid in ascites . A, the left side of the figure shows the fraction of ["C]octanoic acid incorporated in the ascitic chylomicrons as ["C]chylomicron triglyceride (micromoles). B, the right side represents the fraction of [14C]octanoic acid not incorporated in the chylomicrons, present as unesterified fatty acid (micromoles). e-e, patients with cirrhosis and nonchylous ascites; 0 - 0 , patients with cirrhosis and chylous ascites; x x, noncirrhotic patients with chylous ascites.
November 1974
ORIGIN OF FAT IN CHYLOUS ASCITES
In serum. Peak serum octanoic levels occurred between 30 and 60 min after ingestion. As it has been noted before, cirrhotic patients have much higher concentrations than noncirrhotic subjects (peak serum levels: 457 ± 44 J.LM, mean ± SE versus 163 ± 19 J.LM, respectively, P < 0.01) due to shunting and impaired parenchymal function 13 with slower rate of disappearance (5-hr concentration: 94 ± 11 J.LM versus 12 ± 5 J.LM, respectively, P < 0.01) . In all patients, only a small percentage (0 to 6%) of [14 C ]octanoic acid was present in the chylomicron fraction. Discussion This study has demonstrated that most of the ingested triglycerides recovered from chylous ascites were present in the chylomicrons. In the cirrhotic patients, activity of labeled long chain fat found in chylous ascites was much greater (8% of the ingested radioactivity) than in clear ascites (0.6%). An even greater proportion was recovered from that of the noncirrhotic patients (22%) in whom chylous ascites consisted most likely of intestinal lymph. Incorporation of [3H ]oleic acid into ascitic chylomicrons as [3H]chylomicron triglyceride (fig. 2) was significantly higher in the subjects with chylous ascites than in those with clear ascites (P < 0.01). This suggests that intestinal lymph was the main source of chylomicrons in chylous ascites as chylomicrons can permeate intestinal lymphatic capillaries. 22 Chylomicron removal from ascitic fluid was about 30 times slower than removal from blood, as indicated by the respective final decay rates (half-life time: 58 versus 1.8 hr). Nevertheless, it appears that the absolute removal rate from ascites (micromoles per hour) was at least partially dependent on concentrations of chylomicrons in ascitic fluid. Our data also show that the rate of entry of chylomicrons into the ascites was different in three groups, while the removal rate was uninfluenced by disease process (final decay rates were similar), suggesting that the faster rate of appearance and high concentrations of chylomicrons found in chylous ascites were due to a greater leakage of chylomicrons
885
from intestinal lymph into the peritoneal cavity. Removal of the chylomicrons from ascitic fluid by centrifugation completely eliminated the chylous quality of our samples in all instances. The experiments with medium chain fatty acid ( [14 C ]octanoic acid) were designed to study transfer of water-soluble lipid from the vascular compartment into ascites. The absorbed medium chain fatty acid, transported mainly via the portal venous system as free fatty acid, rapidly accumulated in ascitic fluid of both cirrhotic groups. Previous studies have shown that at the concentrations of octanoic acid measured in blood of the cirrhotic patients, a significant portion is free and not bound to protein. 23 Non-protein-bound octanoate, which is water- and fat-soluble, probably diffuses through many membrane systems. It has been shown previously that the non-protein-bound serum fraction of octanoate in the cirrhotic patients is 4 to 5 times higher than normal. Spinal fluid levels were linearly related with, and of the same magnitude of, the free serum octanoate concentration. 23 The same mechanism of rapid diffusion of the non-protein-bound fatty acids from blood into ascites can probably explain the ascitic fluid levels since, in both cirrhotic groups, the concentrations of octanoate not incorporated into the chylomicrons were also 5 times higher as compared with the noncirrhotic patients (fig. 4B). Not all medium chain lipids are transported via the lymphatics as shown by the levels of octanoic acid incorporated in the chylomicrons in ascitic fluid as 14 [ C]chylomicron triglyceride (see fig . 4A), with final decay rates being similar to those of 3H (long chain) chylomicron triglyceride . These results further support the lymphatic origin of chylomicrons in ascitic fluid and suggest that chylous ascites in the cirrhotic originates both from lymph and blood with the chylomicron lipid arising from intestinal lymph and the free fatty acids from blood. Analysis of the composition of chylomicron lipid might provide additional information on the possible mechanism of chylous ascites. Experiments by Fraser 24 have
886
MALAGELADA ET AL.
demonstrated that the size of chylomicrons in lymph is related to the ratios between triglycerides, phospholipids, and cholesterol. When the three main lipid components of ascites are plotted in a triangular system of coordinate (fig. 1), the proportions of the three lipids in cirrhotic patients with chylous ascites appear to be similar to the lymphatic ascites of the noncirrhotic patients (and identical to intestinal lymph), which indicates that the size of the chylomicrons is probably equal. The composition of the lipid fractions in clear ascites, however, is significantly different from chylous ascites, being in the range of the much smaller chylomicrons. As patients with cirrhosis have usually an elevated lymphatic pressure, 25 it is suggested that in all cirrhotic patients with ascites, small chylomicrons escape continuously from lymph capillaries into the ascitic fluid. Chylous ascites in the cirrhotic patient may result from exceptionally high lymphatic pressure leading to rupture of lymph vessels and leakage of whole intestinal lymph into the peritoneal cavity. This assumption is consistent with the anatomical finding of subserosa! intestinallymphagiectasis, recently reported in patients with liver cirrhosis and chylous ascites. 26 REFERENCES 1. Malagelada JR, Iber FL, Linscheer WG: The origin of fat in chylous ascites of patients with liver cirrhosis (abstr). Gastroenterology 62:780, 1972 2. Nix JT, Albert M, Dugas JE, eta! : Chylothorax and chylous ascites: a study of 302 selected cases. Am J Gastroenterol 28 :40-55, 1957 3. Lamotte M, Lamotte-Barrilon, S. Guerre J: L'ascite chyliforme des cirrhotiques. Arch Fr Mal App Dig 57:447-458, 1968 4. Kelley ML, Butt HR: Chylous ascites: an analysis of its etiology. Gastroenterology 39:161-170, 1960 5. Kelley ML, Logan VW: The problem of milky ascites. NY State J Med 61:2307-2315, 1961 6. Waterhouse C, Jaenike JR, Marinetti G: Studies on the nature and origin of pseudochylous ascites. Trans Assoc Am Physicians 71:312-321, 1958 7. Liebowitz HR: Pathogenesis of ascites in cirrhosis of the liver. NY State J Med 69:1895- 2024, 1969 8. Albrink MJ, Glenn WWL, Peters JP, eta!: The transport of lipids in chyle. J Clin . Invest 34:1467-1475, 1955
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9. Morris B, Courtice FC: The origin of chylomicrons in the cervical and hepatic lymph. Q J Exp Physiol 41:341- 348, 1956 10. Courtice FC: The origin of lipoproteins in lymph. In The Lymph and Lymphatic System. Edited by HS Mayerson. Springfield, CC Thomas, 1968, p 119 11. Baker L, Puestow RC, Kruger S, eta!: Estimation of ascitic fluid percent volumes. J Lab Clin Med 39:30-36, 1952 12. Henry RJ, Chiamori N, Ware AG: Determination of bromsulfonphthalein in serum by means of acetone precipitation of proteins. Am J Clin Pathol 32:201- 227, 1959 13. Linscheer WG, Castell DO, Platt RR: A new method for evaluation of portasystemic shunting. The rectal octanoate tolerance test. Gastroenterology 57:415-423, 1969 14. Bray GA: A simple efficient lipid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal Biochem 1:279-285, 1960 15. Alaupovic P, Furman RH, Failor WH, et al: Isolation and characterization of human chyle chylomicrons and lipoproteins. Ann NY Acad Sc 149:791- 807, 1968 16. Noble RP, Campbell FM: Improved accuracy in automated fluorometric determination of plasma triglycerides. Clin Chern 16:166- 170, 1970 17. Henry RJ: Clinical Chemistry. New York, Harper and Row, 1965, p 841-843 18. Total cholesterol procedure (N-24). AutoAnalyzer Manual. Tarrytown, N.Y., 1965 19. Dole VP, Meinertz H: Microdeterminationoflong chain fatty acids in plasma and tissues. J Biol Chern 235:2595-2597, 1960 20. Bartlett RH, Failor WH, Zarafonetis CJD: Lipid studies on human lymph and chyle. JAMA 187:126-127, 1964 21. Lee DS , Hashim SA, Van Itallie TB: Effect of long chain triglyceride on chylous transport of medium chain fatty acids. Am J Physiol 214:294- 297, 1968 22. Courtice FC: The origin of lipoproteins in lymph. In The Lymph and Lymphatic System. Edited by HS Mayerson. Springfield, CC Thomas, 1968, p 98 23. Linscheer WG, Blum AL, Platt RR: Transfer of medium chain fatty acids from blood to spinal fluid in patients with cirrhosis. Gastroenterology 58:509-515, 1970 24. Fraser R: Size and lipid composition of chylomicrons of different Svedberg units of flotation. J Lipid Res 11:60-65, 1970 25. Dumont AE, Mulholland JH: Alterations in thoracic duct lymph flow in hepatic cirrhosis. Ann Surg 156:668- 677, 1962. 26. Caroli J, Ouahnich M: Ascite Chyleuse du cirrhotique. Rev Med Chir Mal Foie 44:99-108, 1969