CASE REPORT

Proteins of Ascitic Fluid in Constrictive Pericarditis S.H. R O B E R T S , MSc, M B B S , MRCP, D.L. K E P K A Y , MD, FRCP(C), and J.A. B A R R O W M A N , BSc, MB, ChB, PhD, M R C P

When ascitic fluid accumulates in patients with chronic hepatic congestion, its protein content is often high, in distinct contrast to low protein levels (usually less than 1.5 g/100 ml) a c c o m p a n y i n g cirrhosis of the liver. The explanation for this difference is not fully understood but observations, both in patients and experimental animals, suggest that elevated hepatic sinusoidal pressure drives plasma across highly permeable sinusoidal m e m branes into the interstitial space of Disse and across the liver capsule. When the capacity of the hepatic lymphatic drainage s y s t e m is exceeded, high protein lymph " w e e p s " into the peritoneal cavity as ascitic fluid of high protein concentration (1, 2). Dive and his colleagues (3) report that the transport of plasma proteins across dog hepatic sinusoids and into hepatic lymph has two major components: molecular sieving of larger proteins and bulk flow. Using their techniques, we studied the transfer of proteins from plasma to ascitic fluid in a patient with gross ascites of high protein concentration resulting from chronic constrictive pericarditis.

CASE REPORT A 17-year-old girl developed secondary amenorrhea in March 1975. Following a negative pregnancy test she was treated unsuccessfully with medroxyprogesterone aceF r o m the D e p a r t m e n t of Medicine, Faculty of Medicine, Memorial University of Newfoundland.

Address for reprint requests: Dr. J.A. Barrowman, Faculty of Medicine, Memorial University, St. John's, Newfoundland, Canada.

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tate. She returned 6 months later with gross ascites. Pelvic examination was unremarkable. Chest x-ray demonstrated small bilateral pleural effusions. The hemoglobin level was 8.6 g/100 ml; the serum bilirubin, LDH, SGOT, and albumin were within normal limits. Abdominal paracentesis failed to reveal malignant cells in the aspirated fluid. Following blood transfusion a laparotomy was performed which revealed the liver to be enlarged and nodular. A wedge biopsy was taken and the ascites was drained. One year later she was readmitted with the additional complaint of easy fatiguability. Despite continued administration of a low-salt diet, spironolactone, and furosemide, the ascites had recurred and persisted. On examination there was an umbilical hernia and massive ascites without peripheral edema. The liver and spleen were not palpable and cardiovascular findings suggested pericardial constriction. The pulse was 80/rain, the blood pressure 100/60 mm Hg lying and standing, and the systolic blood pressure fell 10 mm Hg on deep inspiration. Jugular venous pressure was markedly elevated with little discernible pulsation. A diastolic right ventricular impulse was palpable, and a third heart sound and fine basal pulmonary rales were audible. Investigations at this time revealed linear calcification in the anterior pericardium on chest x-ray; there was no pleural effusion. EKG showed widespread T-wave inversion with right ventricular hypertrophy. Right and left heart catheterization revealed elevation and equalization of the diastolic pressure in all four chambers to 20-25 mm Hg. At this time, hemoglobin, white blood cell count and differential, blood smear, and platelet count were unremarkable. Blood urea nitrogen, bilirubin, alkaline phosphatase, LDH, SGOT, and prothrombin time were also normal. Urinalysis demonstrated a trace of albumin on one examination. Total serum protein was 7.2 g/100 ml and serum albumin 4.4 g/100 ml. Radiologic studies of the small intestine and histologic section of jejunum after

Digestive Diseases, Vol. 23, No. 9 (September 1978) 0002-9211/78/0900-0844505.00/19 1978Digestive Disease Systems, Inc.

ASCITIC FLUID PROTEINS

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Digestive Diseases, Vol. 23, N o . 9 ( S e p t e m b e r 1978)

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ROBERTS ET AL TABLE I. CONCENTRATIONS OF PROTEINS AND PROTEIN-BOUND SUBSTANCES IN SERUM AND ASCITES

Total protein Albumin Transferrin Haptoglobin Ceruloplasmin IgG IgA Ca C4 a-2 macroglobulin IgM Cholesterol Triglycerides Fasting 1V2hr postprandial

Serum

Ascites

Normal serum values (mg/lO0 ml)

7200 4400 358 193 48 1700 300 170 32 250 470 135

5050 2900 170 73 26 975 115 61 14 77 78 65

6000-8000 3450-6500 200-400 60-300 15-60 564-1765 85-385 100-200 20-40 140-344 53-375 150-250

45 95

35 50

30-150

transluminal biopsy were normal without evidence of intestinal lymphangiectasia. Fecal fat excretion (3-day collection) was 0.8 g/day (intake 57 g/day). Barium swallow failed to disclose esophageal varices. Technetium scanning of liver and spleen demonstrated diffuse impairment of uptake in a normal-sized liver. The size of the spleen was at the upper limit of normal. Mantoux test with 10 TU was negative, and urine culture was sterile. Histoplasma complement fixation test was negative. An ascitic fluid aspirate contained mesothelial and occasional mononuclear cells, and its total protein concentration was 5.1 g/100 ml. Routine cultures and cultures for M. tuberculosis were negative. After administration of antituberculous drugs (streptomycin, isoniazid, and ethambutol) thoracotomy revealed a fibrosed and calcified pericardium which was excised with difficulty from the heart and great vessels. Histologic examination showed no inflammatory changes or granulomata while culture of the pericardium for M. tuberculosis was negative at 6 weeks. Postoperatively the jugular venous pressure remained about 11 mm Hg, but ascites reaccumulated. However, with reinstitution of a low-salt diet her abdominal girth decreased and ascites disappeared. A percutaneous liver biopsy was then performed. Liver Histology. The most striking features of the wedge biopsy obtained in September 1975 were dilated sinusoids and blood-filled spaces beneath the capsule, along with extensive fibrosis. Scarring was prominent in portal areas and around groups of hepatocytes producing lobular disarray. Central veins were not visible, and individual hepatocytes were normal. The needle biopsy obtained in November 1976 following relief of the pericardial constriction was studied with both light and electron microscopy. There was extensive sinusoidal dilation and lobular fibrosis which appeared more severe than on the previous biopsy specimen. Sampling differences, however, could have accounted for this apparent change. Individual hepatic cells were isolated by fine fibrosis, and there were mild feathery and hydropic cytoplasmic changes compatible with those seen a few days after major surgery.

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Electron-microscopic studies focused on the space of Disse which was widened in many areas (Figure 1). Extensive deposits of collagen were demonstrated, and these extended between individual liver cells. Amorphous material was also seen but was not further defined. Individual hepatocytes had lost microvilli and granular blebs extended from their rather smooth surface into the space of Disse. In several places a distinct subendothelial basement membrane was present, but it was not continuous. Wide spaces existed between individual sinusoidal cells. Other Investigations. Ten proteins of known molecular weight (albumin, transferrin, haptoglobin, ceruloplasmin, IgG, IgA, IgM, third and fourth component of complement, and a-2 macroglobulin) were measured on single samples of plasma and ascitic fluid by routine methods (Table 1). The ascites-serum concentration ratio was calculated for each protein and compared with the ascitesserum concentration ratio for IgG to minimize uncontrollable causes of variation such as concentration and dilution. IgG was chosen as a reference protein not synthesized by the liver. These values were plotted against the reciprocal of the molecular weight (3) (Figure 2). Plasma and ascitic fluid collected simultaneously were analyzed by molecular exclusion chromatography (Figure 3). DISCUSSION In the presence o f hepatic venous outflow block, both in patients and experimental animals, plasma is driven across highly permeable hepatic sinusoids expanding the interstitial space of Disse (4, 5). Hepatic lymph flow rapidly increases and, w h e n lymphatic drainage capacity is exceeded, interstitial fluid or lymph with high protein concentration " w e e p s " across the liver capsule into the peritoneal cavity (1, 2). The clinical and histologic findings in our patient with constrictive pericarditis suggested that the pathogenesis of ascites was similarly the result of

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ROBERTS ET AL 1.4.

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chronic extreme elevation of hepatic venous pressure. Thoracic lymphatic drainage into the systemic circulation was probably also impeded by the high level of systemic venous pressure (20-25 mm Hg) and possibly by obliteration of intrathoracic lymphatic Channels involved in the pericardial fibrotic process (7). Although other factors could have further c o n t r i b u t e d tO net ascites formation, they would not in this situation account for the high protein concentration of the ascitic fluid. For example, prolonged elevation in systemic venous pressure and extensive portal and perisinusoidal hepatic fibrosis g e n e r a t e d some degree of portal hypertension. Accordingly, transudation of splanchnic filtrate low in protein content, across relatively impermeable intestinal capillaries, could have added to the volume of ascitic fluid (1). This factor was probably of minor importance, however, because ascitic fluid protein levels were high, 5.1 g/100 ml. On the other hand, inflammation and fibrosis involving the peritoneal lymphatic drainage could have impeded the absorption of ascites and contributed directly to formation of high protein fluid. Nonetheless, the absence of inflammatory or neoplastic cells in the ascitic fluid, the lack of evidence for tuberculosis as the cause of pericarditis, and the amelioration of ascites with a reduction in systemic venous pressure rendered these possibilities remote. Digestive Diseases, Vol. 23, No. 9 (September 1978)

D y n a m i c studies of hepatic sinusoidal permeability have not previously been carried out in man. Dive et al (3), however, concluded from experiments in dogs that the transport of serum proteins from hepatic sinusoid to hepatic lymph occurs by two mechanisms. One process corresponds to bulk transfer in which all proteins regardless of size escape from the circulation and pass into lymph, and the other is by molecular sieving where proteins cross the sinusoidai wall at rates governed by size. The two transport components are referred to as "fixed relative permeability coefficient" (FRPC) and "variable relative permeability coefficient" (VRPC), respectively. These coefficients are derived from hepatic lymph-serum concentration ratio of proteins as compared to lymph-serum concentration ratio of a reference protein. Although it may not be strictly correct to use the term "permeability coefficient" in this way, the use of such ratios seems valid as a means of comparing the freedom with which individual proteins traverse the sinusoidal membrane. We therefore used this analysis on the assumption that in our patient ascitic fluid closely resembles and likely originates from hepatic lymph. The regression line (Figure 2) relates the ascitesserum concentration ratios for proteins of various molecular sizes, using the ascites-serum ratio of IgG as a reference. This line does not pass through the origin and thus reflects bulk flow (FRPC). However, molecular exclusion chromatography shows that as the size of each protein moiety increases, progressively smaller concentrations are present in the ascitic fluid (Figure 3). This observation supports the notion of molecular sieving of large protein molecules (FRPC). We believe that hepatic sinusoidal filtration largely accounts for these findings, even

TABLE 2. ASCITES/SERUMCONCENTRATION RATIOS FOR PROTEINS WITH KNOWN MOLECULARWEIGHT Ascites/ serum ratio

Albumin Transferrin Haptoglobin Ceruloplasmin IgG IgA C3 C4 a-2 macroglobin IgM

0.659 0.474 0.378 0.520 0.574 0.383 0.359 0.421 0.308 0.166

A/S ratio protein A / S ratio l g G

Mol. wt (xl06)

1.15 0.83 0.66 0.91 1.00 0.67 0.63 0.73 0.54 0.29

15.4 12.5 10.0 7.6 6.7 6.3 5.5 4.4 1.2 1.1

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ASCITIC FLUID PROTEINS though other pathophysiologic factors, alluded to earlier, probably contributed to ascites formation and may slightly alter the observed protein ratios. Finally, electron-microscopic studies disclosed considerable dilatation of the space of Disse with extensive perisinusoidal collagen deposition and basement membrane formation extending beyond the periportal area where it is normally present (8). "Capillarization" of sinusoids has been noted in cirrhosis, in other fibrotic liver processes (8), as well as with hepatic venous congestion (4). Some consider (9, 10) that the acquired perisinusoidal basement membrane and proliferative collagen network restricts the passage of plasma proteins from hepatic sinusoid to the space of Disse and ultimately into hepatic lymph. If, as the data suggest, the high protein ascites in our patient derives from hepatic lymph, this ultrastructural change does not impair the movement of proteins into and out of hepatic interstitial fluid. SUMMARY A patient with chronic calcific pericarditis, hepatic congestion, and fibrosis had massive ascites with a protein concentration of 5.1 g/100 ml. This fluid was in all likelihood largely derived from hepatic interstitial fluid. The ascites-serum concentration ratio for several protein species and molecular exclusion chromatography of these fluids suggested two processes may be involved in the transfer of protein from serum to ascites, namely bulk transfer of all species and molecular sieving.

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ACKNOWLEDGMENTS

We are indebted to Dr. D. Crosby for permission to study this patient, to Dr. M.H. Gault for biochemical determinations, to Dr. E. Pike for light photomicrographs, Mr. P. Hyam for electron photomicrographs and to Ms C. Mousseau for technical assistance. REFERENCES 1. Witte MH, Witte CL, Dumont AE: Progress in liver disease. Physiological factors involved in the causation of cirrhotic ascites. Gastroenterology 61:742-750, 1971 2. Summerskill WHS, Baldus WP: Ascites. Diseases of the Liver, 4th Edition. L. Schiff(ed). Toronto, J.B. Lippincott, 1975, pp 424-444 3. Dive C, Nadalini AC, Heremans JF: Origin and composition of hepatic lymph proteins in the dog. Lymphology 4:133139, 1971 4. Safran AP, Schaffner F: Chronic passive congestion of the liver in man, electron microscope study of cell atrophy and interlobular fibrosis. Am J Pathol 50:447-463, 1967 5. Bolton C, Barnard WG: The pathological occurrences in the liver in experimental venous stagnation. J Pathol Bacteriol 34:701-709, 1931 6. Hyatt RE, Lawrence G, Smith JR: Observations on the origin of ascites from experimental hepatic congestion. J Lab Clin Med 45:274-280, 1955 7. Servelle M, Bouvrain Y, Tricot R, Soulie J, Turpyn H, Frentz F, Cornu C, Nadim C: Lymphatic circulation in constrictive pericarditis. J Cardiovasc Surg 7:182-200, 1966 8. Schaffner F, Popper H: Electron microscopy of the liver. Diseases of the Liver, 4th Edition. L. Schiff (ed). Toronto, J.B. Lippincott, 1975, pp 51-87 9. Schaffner F, Popper H: Capillarization of hepatic sinusoids in man. Gastroenterology 44:239-242, 1963 10. Dumont AE, Witte CL, Witte MH: Protein content of liver lymph in patients with portal hypertension secondary to hepatic cirrhosis. Lymphology 8:111-113, 1975

Digestive Diseases, Vol. 23, No. 9 (September 1978)

Proteins of ascitic fluid in constrictive pericarditis.

CASE REPORT Proteins of Ascitic Fluid in Constrictive Pericarditis S.H. R O B E R T S , MSc, M B B S , MRCP, D.L. K E P K A Y , MD, FRCP(C), and J.A...
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