[CANCER RESEARCH 32, 1951—1959,September 19721 An Abnormal Orosomucoid in the Plasma of Patients with Neoplastic Disease' Daniel Rudman,2 Perry E. Treadwell, W. Ralph Vogler, Carolyn H. Howard, and Bettye Hollins Departments of Medicine JD. R., W. R. V., C. H. H., B. HI, Biochemistry JD. R./, and Microbiology JP. E. T.J, Emory University School of Medicine, and the ClinicalResearch Facility, Emory University Hospital, A tianta, Georgia 30322 SUMMARY cancer, as it does in those of acute leukemic cases; and (b) to isolate the tryptophan-region material and to determine its Concentrations of the plasma amino acids were measured by chemical structure. As a by-product of this investigation of ion-exchange chromatography in 12 normal subjects (Group tryptophan-region material, further information was accumu I), 10 patients with acute leukemia (Group II), and 38 patients lated on the concentrations of the various plasma amino acids with malignant melanoma or metastatic carcinoma of lung, in patients with neoplastic disease. ovary, or colon (Group III). In Groups II and III, the average concentrations of glutamine, alanine, proline, histidine, and MATERIALS AND METHODS arginine were subnormal. During these analyses, an abnormally elevated peak of ninhydrin-reactive material in the region of Subjects. The subjects studied were 12 normal individuals, the chromatogram ordinarily occupied by tryptophan was ages 23 to 45 (Group I); 10 patients, ages 26 to 53, with acute observed in 75% of the subjects in Groups II and III. However, leukemia (6 lymphocytic and 4 myelocytic) (Group II); and plasma tryptophan concentrations, measured by the specific method of La Du and Michael, were normal. The material 38 patients, ages 26 to 57, with metastatic cancer (Group III). responsible for this peak (“tryptophan-region material―)was The following types of tumor were represented in Group III: isolated by gel-filtration and ion-exchange chromatography. It malignant melanoma (10 patients); bronchogenic carcinoma (9 proved to be a glycoprotein with a molecular weight of 40,000 patients); carcinoma ofthe ovary (10 patients); and carcinoma to 50,000. Its behavior in immunodiffusion, immunoelectro of the colon (9 patients). Individuals were selected for the phoresis, and acrylamide gel electrophoresis at pH 8.9 and 4.0, present study according to the following criteria. First, during and its amino acid composition, were indistinguishable from the period of study, body weight was in the range of ±10%of those of normal human orosomucoid (ct@acid glycoprotein). ideal (calculated according to Metropolitan Life Insurance However, in acrylamide gel electrophoresis at pH 2.7, and in Tables) and did not vary by more than ±1.0 kg/week; food content of hexoses, glucosamine, and sialic acid, the intake amounted to >1200 cal/day (>50 g protein/day). tryptophan-region material isolated from patients in Groups II Second, subjects were afebrile and had no signs of and III differed from normal orosomucoid. Tryptophan-region intercurrent infection when studied. Third, in Groups II material is a form of orosomucoid with a normal protein and III, survival after the analyses reported here was less than moiety but with multiple abnormalities in the carbohydrate I year. Fourthly, in Group III, metastatic involvement of side chains. bone, lung, liver, or lymph nodes had been demonstrated by X-ray or biopsy. Analysis of Plasma Amino Acids. Fasting heparinized blood INTRODUCTION samples were obtained from 7 to 9 a.m. Plasmas were separated within 30 mm and either stored at —20°or We previously described (19) 2 types of abnormality in the processed immediately as specified below. AAN3 was plasma amino acid pattern of most patients with acute measured, on plasma that had been frozen, by the method of leukemia: (a) reduced concentration ofa group of nonessential Frame et a!. (7). Concentrations of individual amino acids amino acids (glutamine, alanine, proline, and histidine); and were measured with lithium citrate buffers by ion-excl@ange (b) a peak of ninhydrin-reactive material in the region of the chromatography in the Beckman 120C instrument, of the 3% ion-exchange chromatogram of the plasma amino acids SSA supernatant fraction offresh plasma (l9).@ Concentration (“aminogram―),ordinarily occupied by tryptophan, but which of tryptophan-region material in plasma is unchanged by other tests showed was not tryptophan. freeze-thawing and by storing at —20°for up to I year. The objectives of the present study were: (a) to learn Plasmas stored at —20°were used, therefore, in most whether tryptophan@region material accumulates in the experiments on isolation and characterization of this material. plasmas of patients with nonleukemic types of advanced Isolation of the Tryptophan-Region Material from Human Plasma. The following procedures were used. For precipitation I This investigation was support@d by USPHS Grants CA 12646-01 3The abbreviations used are: AAN, a-amino nitrogen; SSA, and RR-39. sulfosalicylic acid. 2 To whom requests for reprints should be sent. 4 In selected sera, plasma tryptophan concentration was also Received November 30, 1971; accepted June 2, 1972. measured by the method ofLa Du and Michael(12). SEPTEMBER 1972 1951 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1972 American Association for Cancer Research. Rudman, Treadwell, Vogler, Howard, and Hollins Table 1 Concentrations in plasma ofcertain amino acids (@imoles/liter),tryptophan-region material (HW units /liter), and AAN (mg/J00 ml) Values represent average ±SE. Concentrations that differ from normal (p < 0.05) are footnoted. Concentrations of the following amino acids did not differ significantly among Groups I, II, and III: threonine, serine, aspartic acid, glutamic acid, asparagine, glycine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, and lysine. IIIMalignantBronchogenicCarcinoma Group ofGroup ofCarcinoma colonAminoacid(n IGroup IImelanomacarcinomathe ovarythe =9)Glutamine600 12)(n 10)(n = 10)(n =9)(n=l0)(n 37dProline163 ±54350 ±38°430 ±30b321 ±40C451 ±45435 ± l0'@Alanine315 ±10I 10±11095 ±9c80 ±8'@104 ±II―120 ± 26°Histidine75 ±22167 ±25C250 ±20b181 ±24C195 ±18c200 ± 9°Tryptophanarea2,400± ±347 ±7a55 ±8b10046 ±9d± 600cArginine75 3609,840± 960'@11,900± 830C15,300± 950―13,600± 1010c11,500± @ 8cAAN6.9 ±564 ±838 ±11c43 bc50 ±9°46 ± ±0.085.2 ±0.18―4.9 ±0.26@'5.0 ±o.3oc53 ±0.24@'47 ±0.27c ap < 0.05. b ,, < Cp < 0.005. dp < 0.001. of plasma with SSA, we added to 5 to 20 ml of plasma an (at pH 8.6) (6) were performed against the following antisera equal volume of 6% SSA solution. The mixture was then to human blood fractions, obtained from Hyland and centrifuged at 21,000 X g for 10 mm, and the supernatant Behringwerke Laboratories: rabbit antisera to whole human solution was collected. We then dialyzed various solutions in serum, ct-lipoprotein , transferrin , a2 -haptoglobin , fib rinogen, @ cellophane membranes against distilled H20 for 18 hr at 5°. albumin, 7G-immunoglobulins, Cfi31 A-globulin, ct@-anti Gel filtration of various samples was performed through a 2- x trypsin, a2-HS-glycoprotein, human hemopexin, @32C-glyco 200-cm column of Sephadex G-75 equilibrated with I .0 N protein, and orosomucoid (ct@ acid glycoprotein); horse acetic acid (1 7). “Preparative―5ion-exchangechromatography antisera to fl-lipoprotein and to Gc globulins; and goat was conducted on the short (18-cm) column of the Beckman antiserum to a2 -macroglobulin. In addition, rabbit antisera to 120C amino acid analyzer. The column was programmed for purified samples of tryptophan-region material (labeled analysis of basic amino acids according to Beckman's system “FractionE,―as described under “Results―)wereprepared by for analysis of physiological fluids (22), with the use of injection of this fraction, dissolved in Freund's adjuvant, into sodium citrate buffers. Buffer and temperature changes the rabbit according to the route, dose, and schedule of occurred at 170 mm. Tryptophan eluted at 200 mm. The Campbell et al. (4). sample (5 to 30 mg) was dissolved in 2 ml of sodium citrate The following purified human plasma proteins were buffer, pH 4.5, and was applied to the column. At 185 mm, obtained from Schwarz/Mann Laboratories: albumin, immuno when 154 ml effluent had emerged, the outlet valve was globulin G, fibrinogen, transferrin, and orosomucoid (termed opened into a collecting vessel, and effluent was collected for “commercialorosomucoid―in “Results―). the next 60 mm ; this solution, with a volume of 50 ml, was termed “185-to245-mm fraction.― Characterization of Purified Tryptophan-Region Material. RESULTS The following procedures were used to characterize this material (which proved to be a protein). First, we performed AAN and Aminograms in Patients with Advanced Cancer. acrylamide gel electrophoresis at pH 8.9 , 4.0, and 2 .5 ( I 6). We The level of AAN in the leukemia patients (Group II) and in then measured the amino acid composition (18). Tryptophan the cancer patients (Group III) was significantly lower than content of the protein was determined by the spectrophoto that in the normal subjects (Group 1). Table 1 gives the average metric method of Bencze and Schmid (3). Next, we measured values for each group. In 7 of 10 leukemic cases, and in 30 of the content of hexoses (8), glucosamine, galactosamine [after 38 cancer patients, AAN was more than 2 S.D. below the hydrolysis with 2 N HC1 at 100°for 6 hr under vacuum (22)1, mean of the normals. No gross difference in the frequency of and sialic acid (25). Molecular weight was estimated by gel hypoammnoacidemia in the 4 types of cancer patients was filtration on a calibrated Sephadex G-75 column (17).