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Orosomucoid Content of Pleural and Peritoneal Effusions

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Orosomucoid Content of Pleural and Peritoneal Effusions Orosomucoid Content of Pleural and Peritoneal Effusions Daniel Rudman, … , Elmer C. Hall, Judy M. Conn J Clin Invest. 1974;54(1):147-155. https://doi.org/10.1172/JCI107736. Research Article 22 nonneoplastic, noninflammatory effusions (cirrhosis and congestive heart failure), 12 non-neoplastic inflammatory effusions (tuberculosis, lupus erythematosus, rheumatoid arthritis, and idiopathic pleuropericarditis), and 58 neoplastic effusions (cancer of lung, breast, ovary, and pancreas, and lymphoma) were analyzed by radial immunodiffusion for orosomucoid concentration. The average concentration ±SE was 35±4, 65±17, and 130±13 mg/100 ml in the three types of effusion, respectively. By gel filtration and ion exchange chromatography, orosomucoid was isolated from 12 nonmalignant and 14 malignant fluids. The orosomucoid preparations reacted as single components in acrylamide gel electrophoresis at pH 9.0, and in immunodiffusion and immunoelectrophoresis against antisera to human serum and to human plasma orosomucoid. In radial immunodiffusion, the slope of the line relating concentration to the square of the diameter of the precipitate area was identical for orosomucoid isolated from normal human plasma and from nonneoplastic effusions, but was subnormal for orosomucoid isolated from neoplastic fluids. All orosomucoid preparations had normal amino acid composition. Orosomucoid from the nonmalignant effusions had normal carbohydrate content. 11 of 14 samples of orosomucoid isolated from neoplastic fluids had abnormalities in carbohydrate composition, consisting of subnormal content of sialic acid (11 of 14), hexose (10 of 14), and hexosamine (3 of 14), and abnormally high content of hexosamine (4 of 14). Discriminant analysis showed that concentration of orosomucoid distinguished between neoplastic and nonneoplastic […] Find the latest version: https://jci.me/107736/pdf Orosomucoid Content of Pleural and Peritoneal Effusions DANIEL RUDMAN, RAjENDER K. CHAwLA, ALEJANDRO E. DEL Rio, B1rrYE M. HOLLINS, ELMER C. HALL, and JUDY M. CONN From the Departments of Medicine, Biochemistry, and Biometry, Emory University School of Medicine and the Clinical Research Facility, Emory University Hospital, Atlanta, Georgia 30322 A B S T R A C T 22 nonneoplastic, noninflammatory ef- INTRODUCTION fusions (cirrhosis and congestive heart failure), 12 non- Patients with neoplastic disease frequently have elevated neoplastic inflammatory effusions (tuberculosis, lupus concentration of orosomucoid (al acid glycoprotein) in erythematosus, rheumatoid arthritis, and idiopathic plasma (1). We recently developed a method for iso- pleuropericarditis), and 58 neoplastic effusions (cancer lating homogeneous orosomucoid from as little as 3 ml of lung, breast, ovary, and pancreas, and lymphoma) plasma and found that the circulating orosomucoid in were analyzed by radial immunodiffusion for orosomu- patients with advanced cancer usually possesses an ab- coid concentration. The average concentration ±SE normal carbohydrate composition (2). was 35±4, 65±17, and 130±13 mg/100 ml in the The present study investigated concentration and three types of effusion, respectively. By gel filtration composition of orosomucoid in effusions of neoplastic and ion exchange chromatography, orosomucoid was and nonneoplastic etiology. isolated from 12 nonmalignant and 14 malignant fluids. The orosomucoid preparations reacted as single compo- METHODS nents in acrylamide gel electrophoresis at pH 9.0, and 57 pleural and 35 peritoneal effusions were studied; etiology in immunodiffusion and immunoelectrophoresis against was neoplastic in 58 (Table I). The fluids were anticoagu- antisera to human serum and to human plasma oroso- lated by heparin, centrifuged to remove red blood cells, and stored at -20'C within 1 h after paracentesis. mucoid. In radial immunodiffusion, the slope of the line In all cancer cases, histologic diagnosis and metastatic relating concentration to the square of the diameter of involvement of visceral or parietal pleura or peritoneum the precipitate area was identical for orosomucoid iso- were demonstrated at autopsy. Diagnosis of Laennec's cir- lated from normal human plasma and from nonneoplastic rhosis was confirmed by needle biopsy or autopsy. Each effusion was analyzed for total protein concentra- effusions, but was subnormal for orosomucoid isolated tion (3) and protein electrophoretic pattern (4). In addition, from neoplastic fluids. All orosomucoid preparations 1 ml of each effusion was examined by gel filtration in 1 N had normal amino acid composition. Orosomucoid from acetic acid on a 2 X 200-cm column of Sephadex G-75 (5). the nonmalignant effusions had normal carbohydrate Protein content of the effluent was monitored by measuring optical density at 280 nm. Proteins were recovered from content. 11 of 14 samples of orosomucoid isolated from effluent by lyophilization. neoplastic fluids had abnormalities in carbohydrate com- Homogeneous normal human plasma orosomucoid was position, consisting of subnormal content of sialic acid prepared by purification of commercial human plasma frac- (11 of 14), hexose (10 of 14), and hexosamine (3 of tion VI (Schwarz/Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y.) as described elsewhere (2). Oroso- 14), and abnormally high content of hexosamine (4 of mucoid was isolated from effusions by the method previously 14). developed for plasma (2). To characterize orosomucoid Discriminant analysis showed that concentration of preparations, the following tests were done (2): acrylamide gel electrophoresis at pH 9.0; measurements of amino acid orosomucoid distinguished between neoplastic and non- and carbohydrate compositions; immunodiffusion (at pH neoplastic noninflammatory effusions more effectively 7.2) and immunoelectrophoresis (at pH 8.6) against these than concentration of total protein, albumin, al, fi, antisera (Hyland Div., Travenol Laboratories, Inc., Costa a2, Mesa, Calif., and Behringwerke AG, Marburg-Lahn, W. or a-globulin. Germany): rabbit antisera to whole human serum, oroso- mucoid, a-lipoprotein, transferrin, a2-haptoglobin, fibrinogen, Received for publication 12 Septemnber 1973 and in re- albumin, -yG-immunoglobulin, fl C/Pl1 A-globulin, a1-anti- vised form 8 January 1974. trypsin, a-fetoprotein, a2HS glycoprotein, hemopexin, and The Journal of Clinical Investigation Volume 54 July 1974 147-155 147 TABLE I Protein Analyses Effusion Diagnosis Total Group (case no.) Site protein Albumin ai-globulin a2-globulin g/100 ml g/100 ml g/10O ml g/100 ml I Cirrhosis (1-12) Peritoneal 1.640.25 0.840.14 0.140.02 0.140.02 II Heart failure Pleural 3.4±0.50 1.6±0.25 0.240.03 0.340.04 (13-22) III Carcinoma of the Pleural 2.9±0.29 1.3±0.19 0.4±0.05 0.5±0.07 lung (23-34) IV Lymphoma (35-43) Pleural 2.8±0.42 1.7±0.23 0.140.03 0.3±0.09 V Carcinoma of the Pleural 3.440.34 2.0±0.28 0.0±0.01 0.4±0.05 breast (44-57) VI Carcinoma of the Peritoneal 2.9±0.36 1.5±+0.24 0.2 40.04 0.3±40.04 ovary (58-68) VII Carcinoma of the Peritoneal 3.1±0.30 1.8±0.24 0.2±t0.02 0.3±t0.04 pancreas (69-90) VIII Nonneoplastic, Pleural 3.7 ±0.51 2.24±0.35 0.2 ±0.04 0.3 ±0.12 inflammatory* (91-102) P value, groups (III-I VII) vs. groups (I + II) >0.05 >0.05 >0.05 >0.05 P value, groups (IIII VII) vs. group VIII >0.05 >0.05 >0.05 >0.05 * Tuberculosis, five; lupus erythematosus, three; rheumatoid arthritis, two; idiopathic pleuropericarditis, two. Values represent av±SE. Number of observations is same as number of cases for effusion data; number of plasmas analyzed is indicated in parentheses after total protein value. p2C glycoprotein; horse antisera to P-lipoprotein and Gc 0.3, mol wt 45,000-69,000, representing at least 12 globulins; goat pro- antiserum to at-macroglobulin. Rabbit anti- teins' of in this mol wt c serum to orosomucoid with subnormal carbohydrate con- plasma range. is a prominent tent, isolated from plasma of patient 19 (ref. 2) with acute peak at Ka = 1.0, representing tryptophan, tyrosine, and myelocytic leukemia, was also employed. other substances with mol wt < 3,000 that absorb light Effusions were assayed for content of orosomucoid by at 280 nm. radial immunodiffusion (6) in immunoplates obtained from The elution Behringwerke. profile of 22 nonmalignant effusions (12 In 45% of patients, plasma obtained within 2 days of cirrhotic, 10 congestive heart failure) resembled the paracentesis was analyzed for concentrations of protein (3) normal serum pattern (Fig. 1A and 1B). In contrast, and orosomucoid (6). most neoplastic effusions showed enlargement of peak b RESULTS (Fig. 1C). This peak was recovered by lyophilization, repurified twice by gel filtration on Sephadex G75, and Gel filtration chromatography. The elution profile of analyzed for content of orosomucoid by radial immuno- normal serum (Fig. 1) shows three elevations. These diffusion. Yield of peak b was 10 times greater in neo- peaks, labeled a, b, and c, are identified by their distri- plastic (av 530 mg/100 ml) than in benign effusions bution coefficient, Kd.1 The cube root of Ka is, approxi- (av 58 mg/100 ml); about 20% of this difference was mately, a linear function of square root of the protein's accounted for by increased content of orosomucoid. mol wt (7). a is a major protein peak at Kd 0-0.1, ap- Measurement of orosomucoid in the effusions was proximate mol wt 69,000, representing albumin and most done by the radial immunodiffusion technique (Table plasma globulins. b is a minor protein peak at K4 0.2- 'Prealbumin, orosomucoid, a1-trypsin inhibitor, fetuin, 'Kd= (V. -
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