An Abnormal Orosomucoid in the Plasma of Patients with Neoplastic Disease'

Home , Hemopexin, Orosomucoid, Thyroglobulin

[CANCER RESEARCH 32, 1951â€”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 (â€œtryptophan-region materialâ€•)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 Â±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 Â±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 â€”20Â°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 (â€œaminogramâ€•),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 â€”20Â°for up to I year. The objectives of the present study were: (a) to learn Plasmas stored at â€”20Â°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

Table 1 Concentrations in plasma ofcertain amino acids (@imoles/liter),tryptophan-region material (HW units /liter), and AAN (mg/J00 ml) Values represent average Â±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 Â±54350 Â±38Â°430 Â±30b321 Â±40C451 Â±45435 Â± l0'@Alanine315 Â±10I 10Â±11095 Â±9c80 Â±8'@104 Â±IIâ€•120 Â± 26Â°Histidine75 Â±22167 Â±25C250 Â±20b181 Â±24C195 Â±18c200 Â± 9Â°Tryptophanarea2,400Â± Â±347 Â±7a55 Â±8b10046 Â±9dÂ± 600cArginine75 3609,840Â± 960'@11,900Â± 830C15,300Â± 950â€•13,600Â± 1010c11,500Â± @ 8cAAN6.9 Â±564 Â±838 Â±11c43 bc50 Â±9Â°46 Â± Â±0.085.2 Â±0.18â€•4.9 Â±0.26@'5.0 Â±o.3oc53 Â±0.24@'47 Â±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 5Â°. 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). â€œPreparativeâ€•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 â€œFractionE,â€•as described under â€œResultsâ€•)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 â€œcommercialorosomucoidâ€•in â€œResultsâ€•). the next 60 mm ; this solution, with a volume of 50 ml, was termed â€œ185-to245-mm fraction.â€• 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 100Â°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). apparent. As reported before ( 19), the hypoaminoacidemia of Immunodiffusion (at pH 7.2) (5) and immunoelectrophoresis the leukemic subjects was caused largely by subnormal S The short column, containing PA-35 resin and operated with concentrations of glutamine, alanine, proline, and histidine. sodium citrate buffers, was used in 2 ways: to measure the content of The same plasma amino acids, along with arginine, were â€œtryptophan-region materialâ€• in a sample by automatic ninhydrin analysis of the effluent from the column (â€œninhydrinrunâ€•),and to depleted in Group III. The average degree of depletion of these isolate the material by collecting the effluent of the column at the . 5 amino acids was similar in the 4 types of cancer patient appropriate time intervals (â€œpreparative runâ€•). (Table I).

1952 CANCER RESEARCH VOL. 32

Plasma A minogram in Cancer

Tryptophan-region material was elevated in Groups II and emerged as a single peak in the tryptophan region equivalent III (see Ref. 19 for photographs of this abnormality in the to 6.9 HIV units. Acrylamide gel electrophoresis at pH 8.9 (see tryptophan region of the ammnogram). In normal subjects, the below) showed only I component in this preparation. area [calculated, according to Ref. 22, as height (H) of the Scheme for Isolation of â€œTryptophan-Area Componentâ€• peak multiplied by width (W) at half-heightj of the from Plasma. On the basis of the properties described above, â€œtryptophan region peakâ€•averaged 2,400 11W units/liter of the isolation scheme shown in Chart 2, leading to â€œFraction plasma. In Groups II and III, this value averaged 9,840 and Eâ€•asthe end product,wasadopted.The chromatographic 13,080 HW units/liter, respectively. Significant (more than 2 steps are illustrated in Chart 1. One hundred ml plasma of Case S.D. above the normal average) elevation occurred in 6 of 10 14 were produced in 20-mi batches in this way, yielding a total leukemic patients and in 28 of 38 patients in Group III. In 9 of 180 mg ofFraction E. cancer patients with elevated â€œtryptophan-area peakâ€•in the Characterization of Fraction E from Case 14. One mg of aminogram, a duplicate sample of plasma was analyzed this material was analyzed on the l2OC short and long according to the method of La Du and Michael (12). Plasma columns. Only 1 peak, which was in the tryptophan area and tryptophan was within normal limits(l.0 to 2.1 mg/l00 ml) in which amounted to 7.0 HW units, was observed. On Sephadex all 9 samples. [In the same way, normal tryptophan G-75, a single, symmetrical peak was obtained, with a Kd of concentrations had previously (19) been demonstrated in 0.1 2, corresponding to a molecular weight of about 45,000 leukemic plasmas containing elevated tryptophan region (1 7). Acrylamide gel electrophoresis at pH 8.9 showed I peaks.] component with a mobility rate I .4 times that of albumin Properties of the Tryptophan-Region Material in Plasma of Patients with Acute Leukemia and 4 Types of Advanced A Cancer. One hundred eighty ml plasma with elevated tryptophan region (14,000 11W units/liter) were available through plasmaphoresis of a patient with acute lymphocytic @5o leukemia. Preliminary experiments were done with this plasma to set the stage for an isolation scheme...... 60 First, two 3-mi samples of plasma (each containing 42 units (1)70 @ of the component) were deproteinized with SSA. One sample 80 was analyzed directly on the short ion-exchange column of the I....90 l2OC instrument; the other was dialyzed against water, @ lyophiized, and then placed on the ion-exchange column. In 3 100 such experiments, the recoveries oftryptophan region were 33 0 00 200 300 to 38 11W units and 33 to 36 11W units, respectively. This showed that tryptophan-region material is nondialyzable. VOLUME (ml) A 20-mi sample of the plasma was then deproteinized, and the product of centrifugation was dialyzed and lyophilized, yielding 34 mg of white powder (â€œdialyzed-lyophilized SSA filtrateâ€•). Analysis of 1 mg of this material on the l2OC short B column showed 3.4 HW units of tryptophan region material. Five mg of the dialyzed-lyophiized SSA filtrate (containing I 7 :@:40 HW units) were fractionated by gel filtration on a Sephadex @50 G-75 column. The material emerged in 2 peaks (Chart 1), (I) @60 which were recovered by lyophilization. Analysis of each by chromatography on the short column of the 120C instrument @ (â€œninhydrmnrunâ€•)showed that 90% of the tryptophan-region 80 units placed on the G-75 column had been recovered in the 2nd peak, with a cumulative volume of 2 10 to 270 ml. This I.....90 @ elution position, corresponding to Kd [distribution coefficient 100 of the protein during gel filtration ( 1)] of 0.07 to 0.13, indicated that the molecular weight of the tryptophan-region 0 100 200 300 material was 40,000 to 50,000 (17). VOLUME (ml) Five mg of the dialyzed-lyophilized SSA filtrate were placed

on the short column for a â€œpreparativerun,â€•and the effluent Chart 1 was collected at 185 to 245 mm. This solution was dialyzed, Chart 1. Elution profiles from gel filtration on 2- x 200-cm column lyophilized, reconstituted in 3 ml 1.0 N acetic acid, and placed of Sephadex G-75. Ordinate, cumulative volume of effluent in ml; on a Sephadex G-75 column. The elution profile, consisting abscissa, percentage of transmission ofeffluent at 280 nm. The material essentially of I symmetrical peak at 210 to 270 ml cumulative placed on the column was: A, 5 mg ofdialyzed-Iyophilized SSA filtrate volume, is shown in Chart 1. Effluent at 210 to 270 ml was of plasma; Case 14. B, 3 mg of dialyzed-lyophilized â€œ185- to 245-mmâ€• pooled and lyophilized, yielding 2.0 mg of white powder. One fraction derived from 5 mg of dialyzed-lyophilized SSA filtrate of mg was placed on the l2OC column (nmnhydrin run); it plasma; Case 14.

SEPTEMBER 1972 1953

Plasma human serum showed a single line of precipitation with both protein preparations in imrnunodiffusion and in immunoelec Add equal volume 6%SSA trophoresis; the precipitation line was indistinguishable from that produced by antiorosomucoid. (Fraction A) (Fraction B) One antiserum to Fraction E (Case 14) was raised in the Dialyze; lyophilize (dialyzed rabbit. This antiserum reacted with Fraction E (Case 14) and lyophilized SSA filtrate); gel with normal human commercial orosomucoid in the same way filtration on Sephadex G-75 as did antiorosomucoid (Figs. 2 and 3). Anti-Fraction E (Case column 14) and antiorosomucoid both showed only 1 line of precipitation with human plasma in immunodiffusion and in Effluent at cumulative volume 210 -@270ml; immunoelectrophoresis. This line was more intense with lyophilize (Fraction C) plasmas of patients in Groups II and III, containing elevated tryptophan-area peaks, than with normal plasma (Fig. 3). Preparative ion-exchange Studies on Purified Normal Human Orosomucoid. The chromatography on 120C short column amino acid composition and carbohydrate contents of the commercial orosomucoid preparation differed in some respects Effluent at cumulative volume 154 -@204ml, or from the values reported in the literature. Minor components 185 @245mm(Fraction D) with mobility slower than albumin were evident in acrylamide gel electrophoresis at pH 8 .9, and about 20% of the Dialyze;lyophilize;gel filtration preparation emerged at the void volume of the Sephadex G-75 @ on Sephadex G-75 column column (M.W. 65,000). Therefore, the commercial material was purified in 20-mg @ Effluent at cumulativevolume 210 270 ml; batches according to the flow sheet in Chart 2. The end lyophilize (Fraction E) product (corresponding to Fraction E), obtained in a yield of Chart 2. Flow sheet for isolation of tryptophan-region material from 50 to 70% of the starting material, is listed in Table 2 as plasma. â€œpurified normal human orosomucoid.â€• This preparation emerged from Sephadex G-75 as 1 symmetrical peak with a Kd (Fig. 1). Likewise, at pH 4.0, only 1 band was visualized. of 0.12 (corresponding to a molecular weight of about 45,000) However, at pH 2.7, 5 components were evident (4 of which and migrated as a single component in acrylamide gel at pH were labeled a, b, c, and d for reasons discussed below; the 5th 8.9 and 4.0 (Fig. 1). At pH 2.7, 4 components were seen was a broad band between c and d) (Fig. 1). Amino acid (labeled a, b, c, and d in Fig. 1). Amino acid and carbohydrate analysis of Fraction E (Case 14) gave these results compositions (Table 2) were closely similar to those reported (residues/bOO amino acids): lysine, 76; histidine, 16; arginine, in the literature (9, 11, 20, 26) for normal human 45; aspartic acid, 117; threonine, 87; serine, 49; glutamic acid, orosomucoid. One mg of the material emerged from the short 169; proline, 48; glycine, 40; alanine, 49; half-cystine, 23; column of the Beckman l2OC analyzer as a single peak in the valine, 47; methionine, 7; isoleucine, 53; leucine, 79; tyrosine, tryptophan region with an area of 6.8 to 7.1 HW units. 55 ; phenylalanine, 54; and tryptophan, 19. The carbohydrate On immunodiffusion and immunoelectrophoresis, versus analyses are given in Table 2. Amino acids accounted for about antiorosomucoid and anti-Fraction E (Case 14), purified 60% of the weight of the sample, and carbohydrate accounted normal human orosomucoid was indistinguishable from for about 40%. The amino acid composition was closely commercial normal human orosomucoid and from Fraction E similar to that of human orosomucoid (9, 11, 20, 26), but the (Case 14). carbohydrate composition differed in certain respects. Yields and Properties of Fraction E from the Plasmas of A solution (1 mg/ml) of Fraction E (Case 14) in 0.9% NaCl Other Subjects. Fraction E was now isolated from 5- to 20-mI solution was analyzed in immunodiffusion against 15 specific samples of plasma from 6 normal subjects and from 9 patients antisera to human plasma proteins (as listed in â€œMaterialsand of Groups II and III. The yields from the normals averaged 0.3 Methodsâ€•). Only antiorosomucoid was reactive, a single line of mg/ml plasma. In normal Cases 3 and 5, the amount of precipitation being found. When commercial orosomucoid Fraction E was sufficient for chromatographic, electro from normal human plasma was tested simultaneously, a phoretic, immunological, and chemical study (Table 2; Figs. 1 reaction of identity between Fraction E and the orosomucoid to 3). These preparations were indistinguishable from purified preparation was obtained (Fig. 2). In immunoelectrophoresis, normal orosomucoid in molecular weight (as estimated from antiorosomucoid gave 1 line of precipitation with Fraction E gel filtration), in HW value/mg, in electrophoretic appearance of Case 14 (Fig. 3). This line of precipitation was at pH 8.9, 4.0, and 2.7, and in reactions with antisera to indistinguishable from the reaction of the antiserum with normal orosomucoid and to Fraction E (Case 14). Fraction E commercial normal human orosomucoid. Antisera to normal was obtained in higher yield from the cancer patients (average

Fig. 1. Acrylamkle gel electrophoretograms at pH 8.9 (a) and pH 2.7 (b). Positively charged electrode was below in all instances. a: A , normal serum; B, purified normal human orosomucoid; C, Fraction E from Case 3 (normal);D, Fraction E from Case 14 (acute lymphocytic leukemia);E, Fraction E from Case 24 (malignant melanoma); F, normal human serum. b: A, purified commercial orosomucoid; B to E, Fraction E from neoplastic Cases 14, 19, 24, and 45 (see Table 2 for diagnoses); F, Fraction E from Case 3 (normal).

1954 CANCER RESEARCH VOL. 32

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SEPTEMBER 1972 1955

Table 2 Carbohydrate compositions oforosomucoid preparations Values determined in this study are given as average Â±S.E. (n 3). Each value of Fraction E was compared with the corresponding value of purified normal human orosomucoid. If the p value for this comparison was < 0.05, it was signified according to the symbols used in Table 1.

compositionTryptophan-area Carbohydrate glycoprotemn)aSource mg peak in of corresponding plasmaYield Fraction E(mg/lOO acidWeimeretof preparationDiagnosis(HW units/liter)(mg/ml plasma)Hexoses Glucosamine Sialic

(26)NormalGoaa!. 12.8Schmidetal.(8)Normal16.2 14.0 12.0Heimburgeretal.(20)Normal15.0 15.2 12.1Purified (11)Normal14.7 13.9 0.4orosomucoidFractioncommercialNormal15.5 Â±0.5 15.0 Â±0.6 1 1.9 Â±

ECase 06CaseS3Normal1,9100.315.0 Â±0.7 14.0Â±0.6 13.1Â± 1.2 14.5Â±0.3 12.0Â± 0.3 Case 14 Lymphocytic leukemia 14,000 1.8 188 Â±0.6 9.5 Â±03b I 1.0 Â±0.2 Case 17 Lymphocytic leukemia 9,500 1.3 10.8 Â±0.36 10.7 Â±02b 12.5 Â±0.3 @@ Case 19 Myelocytic leukemia 15,100 2.0 1 1.4 Â±02b 8.7 Â±04b osc @@ Case 24 Malignant melanoma 11,300 1.5 20.0 Â±0â€¢4b 9.1 Â±03b Case 45 Carcinoma ofovary 14,700 1.5 12.0 Â±0.4@' 10.6 Â±O.3'@ 8.0 Â±0.2@' Case03bCase 46Normal Carcinoma ofovary2,050 9,6000.2 0.915.3Â± 14.7 Â±0.3 11.1 Â±07d 16.1 Â± 57 ofcolon Â±0.5@' 14.2 Â±0.3 15.5 Â±O.4'@ Case 32 Bronchogenic carcinoma 15,310 2.1 15.0 Â±0.3 14.4 Â±0.4 15.9 Â±0.4@' Case 36Carcinoma Bronchogenic carcinoma13,950 1 1,5401.6 1.612.2 19.4 Â±0â€¢4b 12.0 Â±02C 7.8 Â±

a Galactosamine was not detectable in purified commercial orosomucoid or in any preparation of Fraction E. b ,, < 0.001. Cp < 0.005. @ d ,@< e ,,<

yield, 1.9 mg/ml plasma). In terms of molecular weight therefore, are characteristics of patients with extensive (estimated by gel filtration), HW value/mg, electrophoretic malignant disease rather than unique features of acute mobility at pH 8.9 and 4.0, and reactions in immunodiffusion leukemia. and immunoelectrophoresis with anti-Fraction E (Case 14) and The tryptophan-region material that accumulates in the antinormal human orosomucoid , these preparations were plasma of patients with neoplastic disease is a form of indistinguishable from normal orosomucoid (purified corn orosomucoid differing from the normal glycoprotein in its mercial variety or that isolated from Cases 3 and 5). In carbohydrate components. By the tests of immunodiffusion acrylamide gel electrophoresis at pH 2.7, however, 6 of the 9 and immunoelectrophoresis at pH 7.2 to 8.6, acrylamide gel preparations of orosomucoid from patients with neoplastic electrophoresis at pH 8.9 and 4.0, amino acid composition, disease differed from the normal preparations: besides Zones and gel-filtration and ion-exchange chromatography, the a, b, c, and d, I or more additional components were visible orosomucoid of the patients in Groups II and III was between the c and d bands (Fig. 1). Amino acid compositions indistinguishable from normal. The differences were detected of the orosomucoids of patients in Groups II and III were in acrylamide gel electrophoresis at pH 2.7 and in the normal, but the contents of hexoses, glucosarnine, and sialic carbohydrate analyses. acid were generally abnormal (Table 2). Glucosamine was Schmid et al. (20) showed that starch gel electrophoresis at reduced in 7 of 9 preparations from leukemia patients or from pH 2.9 resolves normal human orosomucoid into 5 to 8 cancer patients, sialic acid was reduced in 3 and elevated in 3, components; these do not differ in carbohydrate or amino acid and hexoses were subnormal in 4 and elevated in 3. composition, and the structural basis for their differing mobilities is unknown (14, 20). Acrylamide gel electrophoresis at pH 2.7 in the present study resolved normal orosomucoid DISCUSSION into 4 bands. Presumably, these 4 components correspond to some of the 5 to 8 isomers observed by Schmid et al. The same The abnormalities previously observed in AAN and plasma 4 zones were also visible in Fraction E from plasma of patients amino acid chromatograms of patients with acute leukemia with neoplastic disease, but these preparations generally have now been found to occur with similar frequency in each exhibited other bands as well. Evidently, Fraction E from of 4 other types of disseminated malignant disease so far cancer patients is a mixture of several components, some of examined: malignant melanoma, and metastic carcinoma of which may be identical to the isomers of normal human lung, ovary, and colon. Depletion of certain nonessential orosomucoid. How do the remaining constituents of Fraction amino acids and accumulation of tryptophan-region material, E from these patients differ from normal orosomucoid

1956 CANCER RESEARCH VOL. 32

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Fig. 2. Immunodiffusion plates, photographed at 24 hr. a: center well, antiorosomucoid; Well 1, Fraction E (Case 14); Wells 2, 3, and 4, albumin, immunoglobulin G, and transferrin, respectively; Well 5, commercial normal orosomucoid. b: center well, antiserum to Fraction E (Case 14); Well 1, albumin; Well 2, transferrin; Well 3, Fraction E (Case 14); Well4, purified commercial normal orosomucoid; WellS, immunoglobulin G. c: center well, antiorosomucoid; Well 1, empty; Wells2 to 5, Fraction E from Cases 3 (normal), 14 (acute lymphocytic leukemia), 24 (malignant melanoma), and 32 (bronchogenic carcinoma).d: center well, antiserum to Fraction E (Case 14); Wells1 to 5, Fraction E from Cases5 (normal), 14 (acute lymphocytic leukemia), 36 (bronchogenic carcinoma),45 (carcinoma of ovary), and 57 (carcinoma of the colon).

SEPTEMBER 1972 I957

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isomers? Apparently, they do not differ in the protein moiety, The Use of Chicken Antiserum for the Rapid Determination of since the amino acid composition of the entire preparation is Plasma Protein Components. II. The Assay of Human Serum normal; rather, they differ in the carbohydrate side chains. Orosomucoid. J. Lab. Clin. Med., 50: 758â€”768,1957. These consist normally of 5 oligosaccharide units (23) that are 11. Heimburger, N., Heide, K., Haupt, H., and Schultze, H. E. attached to certain amino acid residues of the protein moiety Bauteinanalysen von Humanserumproteinen. Clin. Chim. Acta, 10: by sequential addition of monosaccharides from sugar 293â€”307, 1964. 12. La Du, B. N., and Michael, P. J. An Enzymatic Spectrophotometric nucleotides (23, 24). The irregular pattern of the abnormalities Method for the Determination of Phenylalanine in the Blood. J. in hexose, glucosamine, and sialic acid contents of the Lab. Clin. Med.,55: 491â€”496,1960. orosomucoid preparations from plasmas of Groups II and III 13. MacBeth, R. A., and Bekesi, J. G. Effect ofTransplantable Tumors suggests multiple disturbances in the carbohydrate side chains on the Seromucoid Fraction of Rat Serum. Cancer Res., 24. of this plasma glycoprotein in patients with advanced 2044â€”2051, 1964. neoplastic disease. 14. Marshall,W.E. The Structure ofGlycoproteins. II. The Separation An increased concentration of orosomucoid in cancer and Characterization of the Polymorphic Variants of the a, Acid patients has long been recognized (2, 27), first from qualitative Glycoprotein of Human Serum. J. Biol. Chem., 241: 473 1â€”4737, electrophoretic observations and later from quantitative 1966. immunological measurements. The present gravimetric 15. Molnar, J., Robinson, G. B., and Winzler, R. J. 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Fig. 3. Immunoelectrophoresis plates, photographed at 24 hr. a: Troughs A and B contain antiorosomucoid and antiserum to Fraction E (Case 14), respectively. Wells1, 2, and 3 contain Fraction E (Case 14; acute lymphocytic leukemia), purified commercial normal orosomucoid, and plasma of Case 14, respectively. b: Troughs A and B contain antiorosomucoid and antiserum to Fraction E (Case 14), respectively. Wells1, 2, and 3 contain Fraction E (Case 24; malignant melanoma), purified commercial normal orosomucoid, and normal plasma, respectively.

SEPTEMBER 1972 1959

Daniel Rudman, Perry E. Treadwell, W. Ralph Vogler, et al.

Cancer Res 1972;32:1951-1959.

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