Alejandro E. Del Rio

Novel proteins and peptides in the urine of patients with advanced neoplastic disease

Abstract Gel filtration of human urine on Sephadex ® G-75 columns has revealed that normal subjects excrete 30 to 120 mg. per day of a group of four or more peptides with a molecular weight (MW) of 5,000 to 8,000. These peptides are rich in glycine, proline and alanine, and contain no hydroxyproline; one of the peptides contains over 20 per cent hexose and over 20 per cent hexosamine. This group of MW 5,000 to 8,000 peptides was found to be excreted in 2 to 8 times greater quantity by twenty-seven of fifty-six patients with nineteen different types of disseminated neoplastic disease. In addition, nine of these patients excreted one or more proteins with a molecular weight within the range of 12,000 to 50,000 in concentrations of 3 to 28 mg. per 100 ml. urine. These proteins, smaller in size than plasma albumin, were isolated by gel filtration and characterized according to molecular weight (calculated from distribution coefficient on the Sephadex G-75 column) and electrophoretic mobility. A total of twenty-five proteins was visualized by electrophoresis; only four of them reacted with rabbit antiserum to normal human serum. Four of the twenty-five low molecular weight proteins encountered in the study may have occurred more than once in the urines of different subjects. Thus three patients with metastatic breast cancer excreted a protein with a molecular weight of about 30,000 and mobility 0.5 times that of albumin; two with metastatic carcinoma of the breast excreted a protein with a molecular weight of about 30,000 and mobility 0.8 times that of albumin; and a protein of approximate MW 25,000 and mobility 0.6 times that of albumin was observed in urine samples from one patient with metastatic carcinoma of the breast and by another with metastatic carcinoma of the stomach. Increased excretion of the MW 5,000 to 8,000 group of peptides, and/or excretion of MW 12,000 to 50,000 proteins, was generally found only in the last six months of life (with the exception of two cases of multiple myeloma), whereas in 87 per cent of the cases without these findings the patients were alive six months after study. The data demonstrate that a mild degree of proteinuria in patients with disseminated neoplastic disease frequently represents excretion of proteins in the molecular weight range 12,000 to 50,000, smaller than plasma proteins and perhaps derived from tumor tissue, rather than excretion of plasma proteins through diseased glomeruli.

Orosomucoid Content of Pleural and Peritoneal Effusions

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 noninflammatory effusions more effectively than concentration of total protein, albumin, alpha(1), alpha(2), beta, or gamma-globulin.

Isolation of a novel glycoprotein from the urine of a patient with chronic myelocytic leukemia.

Patient B. J. with chronic myelocytic leukemia excreted 0.5-1.1 g protein per day in the urine. Gel filtration on Sephadex G-75 showed about one-third of this protein to be in molecular weight range 20,000-40,000 (fraction BJC). BJC, prepared from 9 liters of urine by gel filtration, was chromatographed on carboxymethylcellulose. Two proteins were eluted from the resin in pure form (as shown by zone and immunoelectrophoresis) in yields representing 8 and 3 mg/liter of urine: BJC1 and BJC2. Their amino acid compositions were identical. BJC1 contained 61% carbohydrate (33% hexose, 11% sialic acid, 13% glucosamine, 5% galactosamine). BJC2 contained one-fourth to one-half as much of each carbohydrate. Molecular weight of BJC1 was estimated at 29,000 by gel filtration. Neither glycoprotein reacted with rabbit antiserum to normal human serum.Antiserum to BJC1 was made in the rabbit. Immunoelectrophoresis with this antiserum showed a faint precipitin line, corresponding in mobility to BJC1, in normal human plasma, and a stronger line in most leukemic plasmas. By immunodiffusion, BJC1 was not detectable in normal human urine, but a positive reaction occurred in the following conditions: leukemia, 64-72%; other types of disseminated neoplastic disease, 36-78%; regional ileitis, 45%; ulcerative colitis, 38%; tuberculosis, 33%; during the 1st wk after major surgery, 33%.BJC2 was found in the urine by immunoelectrophoresis in 10% of patients with neoplastic disease and was not observed in urine of other patients or in human plasma. Amino acid composition, carbohydrate content, and antigenic specificity indicate BJC1 is a previously unrecognized member of the system of normal human plasma glycoproteins. Like certain other glycoproteins, its plasma concentration frequently increases in patients with neoplastic disease, chronic inflammatory disease, or tuberculosis and after surgery. Because molecular weight is 29,000, increased plasma concentration readily causes its appearance in the urine.